Adeno-associated virus vector

ABSTRACT

Disclosed herein is a recombinant adeno-associated virus (AAV) vector comprising (a) a variant AAV2 capsid protein, wherein the variant AAV2 capsid protein comprises at least four amino acid substitutions with respect to a wild type AAV2 capsid protein; wherein the at least four amino acid substitutions are present at the following positions in an AAV2 capsid protein sequence: 457, 492, 499 and 533; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

FIELD OF THE INVENTION

The present invention relates to the field of recombinant viral vectors.In particular, the invention relates to recombinant viral vectors whichare suitable for the delivery of therapeutic genes in vivo.

BACKGROUND TO THE INVENTION

To date, adeno-associated virus remains one of the most promisingvectors for the delivery of therapeutic genes. A significant number ofpreclinical and clinical studies have firmly established that thisapproach is suitable for the development of gene-based drugs that canreach market approval.

Since the beginning of the development of AAV2 as a vector for genetherapy in the 1980s much progress has been made in optimizing thisplatform for a variety of applications and target tissues. Among thosedevelopments, possibly the most consequential has been the discovery ofa wide variety of serotypes of which ten to twelve are now commonlyexplored. Among the most prominent characteristics of these variousserotypes are their respective relative tissue tropism and—in somecases—the ability of neuronal retrograde transport. Of these serotypes,AAV1-10 are broadly used for pre-clinical and clinical purposes.

A newer platform has been developed that involves processes that allowfor the targeting and de-targeting of specific tissues and cellsub-types in patients. The core technology of these approaches is basedon trial and error evaluation of existing AAV variants (serotypes) andin vivo selection of randomly introduced AAV capsid mutants. Together,these two promising approaches provide tens—if not hundreds of potentialvectors with different transduction behaviour.

The most intriguing aspect of AAV serotypes is their ability toefficiently transduce specific tissues in animal models and in man. Todate, comprehensive molecular understanding of the underlying mechanismsfor the tissue tropism has yet to be put forward and it is thusgenerally assumed that the available tissue-specific receptors for eachserotype play a central role in the efficient transduction by thevarious serotypes.

Accordingly there is still a need for additional AAV vectors, which haveimproved properties in terms of in vivo transgene expression and tissuespecificity. In particular, such vectors have the potential to providegreatly enhanced benefits for gene delivery to various target tissues inhumans.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a recombinantadeno-associated virus (AAV) vector comprising: (a) a variant AAV2capsid protein, wherein the variant AAV2 capsid protein comprises atleast four amino acid substitutions with respect to a wild type AAV2capsid protein; wherein the at least four amino acid substitutions arepresent at the following positions in an AAV2 capsid protein sequence:457, 492, 499 and 533; and (b) a heterologous nucleic acid comprising anucleotide sequence encoding a gene product.

In one embodiment, the variant AAV capsid protein comprises a sequenceof SEQ ID NO:2, or a sequence having at least 95% sequence identitythereto. In another embodiment, the wild type AAV capsid proteincomprises a sequence of SEQ ID NO:1.

In one embodiment, the variant AAV2 capsid protein comprises one or moreof the following residues: M457, A492, D499 and Y533. In a preferredembodiment, the variant AAV2 capsid protein comprises one or more of thefollowing amino acid substitutions with respect to a wild type AAV2capsid protein: Q457M, S492A, E499D and F533Y.

In one embodiment, the variant AAV2 capsid protein further comprises oneor more amino acid substitutions with respect to the wild type AAVcapsid protein at the following positions in the AAV2 capsid proteinsequence: 125, 151, 162 and 205. In a preferred embodiment, the variantAAV2 capsid protein comprises one or more of one or more of thefollowing residues: I125, A151, S162 and S205. In another preferredembodiment, the variant AAV2 capsid protein comprises one or more of thefollowing amino acid substitutions with respect to a wild type AAV2capsid protein: V125I, V151A, A162S and T205S.

In one embodiment, the variant AAV2 capsid protein further comprises oneor more amino acid substitutions with respect to the wild type AAVcapsid protein at the following positions in the AAV2 capsid proteinsequence: 585 and 588. Preferably the variant AAV2 capsid proteincomprises one or more of one or more of the following residues: S585 andT588. More preferably the variant AAV2 capsid protein comprises one ormore of the following amino acid substitutions with respect to a wildtype AAV2 capsid protein: R585S and R588T.

In one embodiment, the variant AAV2 capsid protein further comprises oneor more amino acid substitutions with respect to the wild type AAVcapsid protein at the following positions in the AAV2 capsid proteinsequence: 546, 548 and 593. Preferably the variant AAV2 capsid proteincomprises one or more of one or more of the following residues: D546,G548, and S593. More preferably the variant AAV2 capsid proteincomprises one or more of the following amino acid substitutions withrespect to a wild type AAV2 capsid protein: G546D, E548G and A593S.

In one embodiment, the variant AAV2 capsid protein comprises the residueN312, i.e. the residue which is present in the wild type AAV2 capsidprotein at position 312. In this embodiment, the variant AAV2 capsidprotein is not mutated at position 312 compared to the wild type AAV2capsid protein sequence.

In another aspect, the present invention provides a recombinantadeno-associated virus (AAV) vector comprising: (a) a variant AAV8capsid protein, wherein the variant AAV8 capsid protein comprises anamino acid substitution with respect to a wild type AAV8 capsid proteinat position 315 in an AAV8 capsid protein sequence; and (b) aheterologous nucleic acid comprising a nucleotide sequence encoding agene product.

In one embodiment, the variant AAV capsid protein comprises a sequencehaving at least 95% sequence identity to SEQ ID NO:6. In anotherembodiment, the wild type AAV capsid protein comprises a sequence of SEQID NO:6.

In one embodiment, the variant AAV8 capsid protein comprises the aminoacid substitution S315N with respect to a wild type AAV8 capsid protein.Preferably the AAV8 capsid protein sequence comprises one or more aminoacid substitution present at one or more of the following positions:125, 151, 163, 206, 460, 495, 502, 536, 549, 551, 588, 591 and/or 596.

In a preferred embodiment, the variant AAV8 capsid protein comprises oneor more of the following amino acid substitutions with respect to a wildtype AAV8 capsid protein: (a) V125I, Q151A, K163S, A206S, T460M, T495A,N502D, F536Y, N549D, A551G, Q588S and/or G596S; and/or (b) T591R.

In another aspect, the present invention provides a recombinantadeno-associated virus (AAV) vector comprising: (a) a variant AAV3Bcapsid protein, wherein the variant AAV3B capsid protein comprises anamino acid substitution with respect to a wild type AAV3B capsid proteinat position 312 in an AAV3B capsid protein sequence; and (b) aheterologous nucleic acid comprising a nucleotide sequence encoding agene product.

In one embodiment, the variant AAV3B capsid protein comprises a sequencehaving at least 95% sequence identity to SEQ ID NO:11. In anotherembodiment, the wild type AAV capsid protein comprises a sequence of SEQID NO:11.

In one embodiment, the variant AAV3B capsid protein comprises the aminoacid substitution S312N with respect to a wild type AAV3B capsidprotein.

In another aspect, the present invention provides a recombinantadeno-associated virus (AAV) vector comprising (a) a variant AAV-LK03capsid protein, wherein the variant AAV-LK03 capsid protein comprises anamino acid substitution at position 312 with respect to a AAV-LK03capsid protein sequence as defined in SEQ ID NO:12; and (b) aheterologous nucleic acid comprising a nucleotide sequence encoding agene product.

In one embodiment, the variant AAV-LK03 capsid protein comprises asequence having at least 95% sequence identity to SEQ ID NO:12.

In another aspect, the present invention provides a recombinantadeno-associated virus (AAV) vector comprising: (a) a variant AAV capsidprotein, wherein the variant AAV capsid protein comprises at least oneamino acid substitution with respect to a wild type AAV capsid proteinat a position corresponding to one or more of the following positions inan AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499,533, 546, 548, 585, 588 and/or 593; and (b) a heterologous nucleic acidcomprising a nucleotide sequence encoding a gene product.

In one embodiment, the at least one amino acid substitution is presentat one or more of the following positions in an AAV2 capsid proteinsequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585,588 and/or 593; or at one or more corresponding positions in analternative AAV capsid protein sequence.

In one embodiment, the vector comprises a variant AAV2 capsid protein.In another embodiment, the variant AAV capsid protein comprises asequence of SEQ ID NO:2, or a sequence having at least 95% sequenceidentity thereto. In another embodiment, the wild type AAV capsidprotein is from AAV2. In another embodiment, the wild type AAV capsidprotein comprises a sequence of SEQ ID NO:1.

In one embodiment, the variant AAV2 capsid protein comprises one or moreof the following residues: I125, A151, S162, S205, S312, M457, A492,D499, Y533, D546, G548, S585, T588 and/or S593. In a preferredembodiment, the variant AAV2 capsid protein comprises one or more of thefollowing amino acid substitutions with respect to a wild type AAV2capsid protein: V125I, V151A, A162S, T205S, N312S, Q457M, S492A, E499D,F533Y, G546D, E548G, R585S, R588T and/or A593S.

In further embodiments, the variant AAV capsid protein is from AAV1,AAV5, AAV6, AAV8, AAV9 or AAV10.

In one embodiment, the vector comprises a variant AAV1 capsid protein.In another embodiment, the variant AAV capsid protein comprises asequence having at least 95% sequence identity to SEQ ID NO:3. Inanother embodiment, the wild type AAV capsid protein is from AAV1. Inanother embodiment, the wild type AAV capsid protein comprises asequence of SEQ ID NO:3.

In one embodiment, at least one amino acid substitution is present atone or more of the following positions in the AAV1 capsid proteinsequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586,589 and/or 594. In a preferred embodiment, the variant AAV1 capsidprotein comprises one or more of the following amino acid substitutionswith respect to a wild type AAV1 capsid protein: V125I, Q151A, T162S,N313S, N458M, K493A, N500D, F534Y, S547D, and/or G594S. In analternative embodiment, the variant AAV1 capsid protein comprises one ormore of the following amino acid substitutions with respect to a wildtype AAV1 capsid protein: S205T, G549E, S586R and/or T589R.

In one embodiment, the vector comprises a variant AAV5 capsid protein.In another embodiment, the variant AAV capsid protein comprises asequence having at least 95% sequence identity to SEQ ID NO:4. Inanother embodiment, the wild type AAV capsid protein is from AAV5. Inanother embodiment, the wild type AAV capsid protein comprises asequence of SEQ ID NO:4.

In one embodiment, at least one amino acid substitution is present atone or more of the following positions in the AAV5 capsid proteinsequence: 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575,578 and/or 583. In a preferred embodiment, the variant AAV5 capsidprotein comprises one or more of the following amino acid substitutionswith respect to a wild type AAV5 capsid protein: V124I, K150A, K153S,A195S, R303S, T444M, S479A, V486D, T520Y, P533D, and/or G583S. In analternative embodiment, the variant AAV5 capsid protein comprises one ormore of the following amino acid substitutions with respect to a wildtype AAV5 capsid protein: G537E, S575R and/or T578R.

In one embodiment, the vector comprises a variant AAV6 capsid protein.In another embodiment, the variant AAV capsid protein comprises asequence having at least 95% sequence identity to SEQ ID NO:5. Inanother embodiment, the wild type AAV capsid protein is from AAV6. Inanother embodiment, the wild type AAV capsid protein comprises asequence of SEQ ID NO:5.

In one embodiment, at least one amino acid substitution is present atone or more of the following positions in the AAV6 capsid proteinsequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586,589 and/or 594. In a preferred embodiment, the variant AAV6 capsidprotein comprises one or more of the following amino acid substitutionswith respect to a wild type AAV6 capsid protein: V125I, Q151A, T162D,N313S, N458M, K493A, N500D, F534Y, S547D, and/or G594S. In analternative embodiment, the variant AAV6 capsid protein comprises one ormore of the following amino acid substitutions with respect to a wildtype AAV6 capsid protein: S205T, G549E, S586R and/or T589R.

In one embodiment, the vector comprises a variant AAV8 capsid protein.In another embodiment, the variant AAV capsid protein comprises asequence having at least 95% sequence identity to SEQ ID NO:6. Inanother embodiment, the wild type AAV capsid protein is from AAV8. Inanother embodiment, the wild type AAV capsid protein comprises asequence of SEQ ID NO:6.

In one embodiment, at least one amino acid substitution is present atone or more of the following positions in the AAV8 capsid proteinsequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588,591 and/or 596. In a preferred embodiment, the variant AAV8 capsidprotein comprises one or more of the following amino acid substitutionswith respect to a wild type AAV8 capsid protein: V125I, Q151A, K163S,A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and/or G596S. Inan alternative embodiment, the variant AAV8 capsid protein comprises oneor more of the following amino acid substitutions with respect to a wildtype AAV8 capsid protein: S315N and/or T591R.

In one embodiment, the vector comprises a variant AAV9 capsid protein.In another embodiment, the variant AAV capsid protein comprises asequence having at least 95% sequence identity to SEQ ID NO:7. Inanother embodiment, the wild type AAV capsid protein is from AAV9. Inanother embodiment, the wild type AAV capsid protein comprises asequence of SEQ ID NO:7.

In one embodiment, at least one amino acid substitution is present atone or more of the following positions in the AAV9 capsid proteinsequence: 125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586,589 and/or 594. In a preferred embodiment, the variant AAV9 capsidprotein comprises one or more of the following amino acid substitutionswith respect to a wild type AAV9 capsid protein: L125I, Q151A, N314S,Q458M, V493A, E500D, F534Y, G547D, A589T and/or G594S. In an alternativeembodiment, the variant AAV9 capsid protein comprises one or more of thefollowing amino acid substitutions with respect to a wild type AAV9capsid protein: S162A, S205T, G549E and/or S586R.

In one embodiment, the vector comprises a variant AAV10 capsid protein.In another embodiment, the variant AAV capsid protein comprises asequence having at least 95% sequence identity to SEQ ID NO:8. Inanother embodiment, the wild type AAV capsid protein is from AAV10. Inanother embodiment, the wild type AAV capsid protein comprises asequence of SEQ ID NO:8.

In one embodiment, at least one amino acid substitution is present atone or more of the following positions in the AAV10 capsid proteinsequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588,591 and/or 596. In a preferred embodiment, the variant AAV10 capsidprotein comprises one or more of the following amino acid substitutionswith respect to a wild type AAV10 capsid protein: V125I, Q151A, K163S,A206S, N315S, T460M, L495A, N502D, F536Y, G549D, Q588S, A591T and/orG596S. In an alternative embodiment, the variant AAV10 capsid proteincomprises the following amino acid substitution with respect to a wildtype AAV10 capsid protein: G551E.

In one embodiment, the recombinant AAV vector exhibits increasedtransduction of a neuronal or retinal tissue compared to an AAV vectorcomprising a corresponding wild type AAV capsid protein.

In another embodiment, the recombinant AAV vector exhibits increasedtransduction of liver tissue compared to a corresponding wild type AAVcapsid protein.

In one embodiment, the gene product comprises an interfering RNA or anaptamer. In another embodiment, the gene product comprises apolypeptide. Preferably the gene product comprises a neuroprotectivepolypeptide, an anti-angiogenic polypeptide, or a polypeptide thatenhances function of a neuronal or retinal cell. In preferredembodiments, the gene product comprises glial derived neurotrophicfactor, fibroblast growth factor, nerve growth factor, brain derivedneurotrophic factor, rhodopsin, retinoschisin, RPE65 or peripherin.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: (a) a recombinant AAV vector as defined above;and (b) a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a method fordelivering a gene product to a tissue in a subject, the methodcomprising administering to the subject a recombinant AAV vector orpharmaceutical composition as defined above.

In some embodiments, the tissue is selected from blood, bone marrow,muscle tissue, neuronal tissue, retinal tissue, pancreatic tissue, livertissue, kidney tissue, lung tissue, intestinal tissue or heart tissue.Preferably the tissue is neuronal, retinal or liver tissue.

In another aspect, the present invention provides a method for treatinga disorder in a subject, the method comprising administering to thesubject a recombinant AAV vector or pharmaceutical composition asdefined above. In some embodiments, the disorder is a neurological,ocular or hepatic disorder.

In another aspect, the present invention provides a recombinant AAVvector or pharmaceutical composition as defined above, for use intreating a disorder in a subject. In some embodiments, the disorder is aneurological, ocular or hepatic disorder. Preferably the neurologicaldisorder is a neurodegenerative disease. In an alternative embodiment,the ocular disorder is glaucoma, retinitis pigmentosa, maculardegeneration, retinoschisis or diabetic retinopathy.

In another aspect, the present invention provides an isolated variantAAV capsid protein, wherein the variant AAV capsid protein comprises atleast one amino acid substitution with respect to a wild type AAV capsidprotein; wherein the at least one amino acid substitution is present atone or more of the following positions in an AAV2 capsid proteinsequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585,588 and/or 593; or at one or more corresponding positions in analternative AAV capsid protein sequence.

In another aspect, the present invention provides an isolated nucleicacid comprising a nucleotide sequence that encodes a variant AAV capsidprotein as defined above.

In another aspect, the present invention provides an isolated host cellcomprising a nucleic acid as defined above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of wild-type adeno-associated virus2 capsid protein VP1 (SEQ ID NO:1; NCBI Reference Sequence: NC_001401).Residues V125, V151, A162, T205, N312, Q457, S492, E499, F533, G546,E548, R585, R588 and A593 are highlighted.

FIG. 2 shows the amino acid sequence of true-type adeno-associated virus2 (ttAAV2) capsid protein VP1 (SEQ ID NO:2). Residues I125, A151, S162,S205, S312, M457, A492, D499, Y533, D546, G548, S585, T588, S593 differcompared to wild-type AAV2 VP1 (SEQ ID NO:1) and are highlighted.

FIG. 3 shows the amino acid sequence of wild-type adeno-associated virus1 capsid protein VP1 (SEQ ID NO:3; NCBI Reference Sequence: NC_002077).Highlighted residues: S205 (aligns with S205 in ttAAV2 (SEQ IDNO:2))-G549 (aligns with G548 in ttAAV2)-S586 (aligns with S585 inttAAV2)-T589 (aligns with T588 in ttAAV2).

FIG. 4 shows the amino acid sequence of wild-type adeno-associated virus5 capsid protein VP1 (SEQ ID NO:4; NCBI Reference Sequence: AF085716).Highlighted residues: G537 (aligns with G548 in ttAAV2)-S575 (alignswith S585 in ttAAV2)-T578 (aligns with T588 in ttAAV2).

FIG. 5 shows the amino acid sequence of wild-type adeno-associated virus6 capsid protein VP1 (SEQ ID NO:5; NCBI Reference Sequence: AF028704).Highlighted residues: S205 (aligns with S205 in ttAAV2)-G549 (alignswith G548 in ttAAV2)-S586 (aligns with S585 in ttAAV2)-T589 (aligns withT588 in ttAAV2).

FIG. 6 shows the amino acid sequence of wild-type adeno-associated virus8 capsid protein VP1 (SEQ ID NO:6; NCBI Reference Sequence: NC_006261).Highlighted residues: S315 (aligns with S312 in ttAAV2)-T591 (alignswith T588 in ttAAV2).

FIG. 7 shows the amino acid sequence of wild-type adeno-associated virus9 capsid protein VP1 (SEQ ID NO:7; NCBI Reference Sequence: AY530579).Highlighted residues: S162 (aligns with S162 in ttAAV2)-S205 (alignswith S205 in ttAAV2)-G549 (aligns with G548 in ttAAV2)-S586 (aligns withS585 in ttAAV2).

FIG. 8 shows the amino acid sequence of wild-type adeno-associated virus10 capsid protein VP1 (SEQ ID NO:8). Highlighted residue: G551 (alignswith G548 in ttAAV2).

FIG. 9 shows an alignment of AAV capsid protein VP1 amino acidsequences.

FIG. 10 The plasmid used to produce AAV2 vectors was the packagingplasmid pDG. Above: pDG with the wild-type AAV2 genes. Below: pDG-ttAAV2with the true-type AAV2 genes, highlighted are the two key mutations inthe heparan binding domains at positions 585 and 588. MMTV: promoterdriving AAV rep expression, E2a, E4ORF6 and VA are the genes expressingadenovirus helper factors.

FIG. 11 Quantification of viral titres of rAAV2 true-type (TT) andwild-type (WT) for in vivo injections by SDS-PAGE showing Kryptonstaining for separated proteins, and scanned using aninfrared-fluorescence scanner (Odyssey Imaging systems). A: 10 μlof AAV2virus particles, and 62.5 ng-500 ng of BSA were separated on a 12%separating gel containing SDS and stained with Krypton Protein Stain.The image was converted to grayscale. The capsid gene proteins VP1, VP2,VP 3 are labelled on the left. B: Table showing titres from qPCR (vectorgenome [vg/ml]) and SDS-Page (capsid titre [capsid/ml]).

FIG. 12 A. Representative examples of rat brain sections stained with aGFP-specific antibody are shown. The vector was injected into thestriatum as shown by the arrow. B. representative example of aninjection into the substantia nigra is shown.

FIG. 13 GFP transduction of the eye using ttAAV2 and wtAAV2 is shown. A.Retina in a transverse section is shown after ttAAV2 (top) and wtAAV2(bottom) vector administration is shown. B. Magnifications of the dashedboxes in A are shown.

FIG. 14 Transduction of mouse brains after neonatal vector injection.i.v., intra-venous vector administration; i.c., intra-cranial injection;AAV-2, wtAAV2; AAV-TT, ttAAV2.

FIG. 15 Three-dimensional representation of the AAV2 capsid. Thehighlighted residues correspond to the amino acid changes between ttAAV2and wild-type particles, grouped by colour depending on their position.

FIG. 16 Representation of a threefold spike on the AAV2 capsid. Thehighlighted residues correspond to the amino acid changes betweenTrue-type and Wild-type particles. The heparin binding site residues arehighlighted in green.

FIG. 17 Representation of the internal side of the AAV2 capsid. Thehighlighted residues in light-blue correspond to the single amino acidchange in ttAAV2 that is located on the internal side of the capsid.

FIG. 18 Representation of a threefold spike on the AAV2 capsid. Theresidues highlighted in beige correspond to two amino acid changes inthe True-type vector that are spatially close and located in the groovebetween two threefold-proximal peaks on the AAV capsid.

FIG. 19 Representation of a threefold spike on the AAV2 capsid. Theresidue highlighted in brown corresponds to a single isolated amino acidchange (S593) in the True-type vector that is located in the groovebetween threefold-proximal peaks

FIG. 20 Representation of a threefold spike on the AAV2 capsid. The fouramino acids highlighted in pink are involved in receptor binding andclosely situated on the threefold spikes.

FIG. 21 Three-dimensional representation of an alignment between VP1capid monomer from AAV2 (light blue) and VP1 monomer from AAV1 (orange).The highlighted residues in the middle-left of the picture correspond toG549 in AAV1 (orange spheres) and E548 in AAV2 (cyan sphere). Thehighlighted residues in the top-right of the picture correspond to S586and T589 in AAV1 (orange spheres) and R585 and R588 in AAV2 (cyansphere).

FIG. 22 Three-dimensional representation of an alignment between VP1capsid monomer from AAV2 (light blue) and VP1 monomer from AAV5(purple). The highlighted residues in the middle of the picturecorrespond to G537 in AAV5 (purple spheres) and E548 in AAV2 (cyansphere). The highlighted residues in the top-right of the picturecorrespond to S575 and T578 in AAV5 (purple spheres) and R585 and R588in AAV2 (cyan sphere).

FIG. 23 Three-dimensional representation of an alignment between VP1capsid monomer from AAV2 (light blue) and VP1 monomer from AAV6(yellow). The highlighted residues in the bottom of the picturecorrespond to G549 in AAV6 (orange spheres) and E548 in AAV2 (cyansphere). The highlighted residues in the top-right of the picturecorrespond to S586 and T589 in AAV6 (orange spheres) and R585 and R588in AAV2 (cyan sphere).

FIG. 24 Three-dimensional representation of an alignment between VP1capsid monomer from AAV2 (light blue) and VP1 monomer from AAV8 (pink).The highlighted residues in the top-left of the picture correspond toS315 in AAV8 (red spheres) and N312 in AAV2 (cyan sphere). Thehighlighted residues in the bottom-right of the picture correspond toT591 in AAV8 (red spheres) and R588 in AAV2 (cyan sphere).

FIG. 25 Three-dimensional representation of an alignment between VP1capsid monomer from AAV2 (light blue) and VP1 monomer from AAV9 (green).The highlighted residues in the middle of the picture correspond to G549in AAV9 (yellow spheres) and E548 in AAV2 (cyan sphere). The highlightedresidues in the bottom-left of the picture correspond to S586 in AAV9(yellow spheres) and R585 in AAV2 (cyan sphere).

FIG. 26 Analysis of rAAV2 TT and WT expression in the parafascicularisnucleus after striatal injection in rat brain. A: Representative imagesof rat brain sections showing the rostral side on the left and thecaudal side on the right. The site of injection in the striatum isindicated, and the area of projection in the hypothalamus observed in Band C is shown (parafascicularis nucleus, pf). B and C: Highmagnification images of the GFP expression detected in theparafascicularis nucleus (pf) after striatal injection of rAAV2 WT (B)or TT (C).

FIG. 27 Overview of intracranial injections of rAAV2 TT and WT inneonatal mice. Representative examples of neonate brain sections stainedwith a GFP-specific antibody are shown. 5×10¹⁰ vg of rAAV2 TT (top) orrAAV2 WT (middle) were injected into the lateral ventricle of neonatalmouse brains. An uninjected brain from a neonatal mouse, stainedsimultaneously, is represented as a negative control (NT, nontransduced).

FIG. 28 High magnification pictures of neonatal mouse brain sectionsafter intracranial injections of rAAV2 TT or WT. Neonate brain sectionsstained with a GFP-specific antibody are shown. 5×10¹⁰ vg of rAAV2 TT(left panels) or rAAV2 WT (right panels) were injected into the lateralventricle of neonatal mouse brains. S1BF: barrel field primarysomatosensory cortex.

FIG. 29 Overview of brain transduction after systemic injection of rAAV2TT and WT in neonatal mice. Representative examples of neonate brainsections stained with a GFP-specific antibody are shown. 2×10¹¹ vg ofrAAV2 TT (top) or rAAV2 WT (bottom) were injected into the jugular veinsof neonatal mice.

FIG. 30 High magnification pictures of neonatal mouse brain sectionsafter systemic injections of rAAV2 TT or WT. Neonate brain sectionsstained with a GFP-specific antibody are shown. 2×10¹¹ vg of rAAV2 TT(left panels) or rAAV2 WT (right panels) were injected into the jugularveins of neonatal mice. S1BF: barrel field primary somatosensory cortex.

FIG. 31 High magnification pictures of neonatal mouse tissue sectionsafter systemic injections of rAAV2 TT or WT. 2×10¹¹ vg of rAAV2 TT orrAAV2 WT were injected into the jugular veins of neonatal mice.Uninjected mouse organs were used as negative controls.

FIG. 32 High magnification images of adult rat brain sections afterstriatal injections of rAAV2 TT, WT and HBnull. Representative examplesof rat brain sections stained with a GFP-specific antibody are shown.3.5×10⁹ vg of rAAV2 WT (left), TT (right) or AAV2-HBnull (middle) wereinjected into the striatum of adult rat brains and representativepictures were taken in the thalamus or in the substantia nigra (SN).

FIG. 33 Overview of intracranial injections of the full AAV-TT comparedwith various TT mutants in neonatal mice. Representative examples ofneonate brain sections stained with a GFP-specific antibody are shown.5×10¹⁰ vg of rAAV2 TT, TT-S312N, TT-S593A or TT-D546G/G548E (TT-DG) wereinjected into the lateral ventricle of neonatal mouse brains. Anuninjected brain from a neonatal mouse, stained simultaneously, isrepresented as a negative control (NT).

FIG. 34 High magnification pictures of neonatal mouse brain sectionsafter intracranial injections of various TT mutant vectors. Neonatebrain sections stained with a GFP-specific antibody are shown. 5×10¹⁰ vgof vectors were injected into the lateral ventricle of neonatal mousebrains. TT-DG: TT-D546G/G548E.

FIG. 35 Overview of neonatal mice intracranial injections of the fullAAV-TT compared with the TT-S312N mutant and the potential final TTvector containing 10 mutations. Representative examples of neonate brainsections stained with a GFP-specific antibody are shown. 5×10⁰⁹ vg ofrAAV2 TT, TT-S312N, TT or TT-S312N-D546G/G548E-S593A (TT-S312N-DG-S593A)were injected into the lateral ventricle of neonatal mouse brains. Anuninjected brain from a neonatal mouse, stained simultaneously, isrepresented as a negative control (NT).

FIG. 36 High magnification pictures of neonatal mouse brain sectionsafter intracranial injections of various TT mutant vectors. Neonatebrain sections stained with a GFP-specific antibody are shown. 5×10⁰⁹ vgof vectors were injected into the lateral ventricle of neonatal mousebrains.

FIG. 37 ELISA quantification of GFP protein in neonatal mice brainsinjected with the full AAV-TT, the TT-S312N mutant or theTT-S312N-DG-S593A. 5×10⁰⁹ vg of vectors were injected into the lateralventricle of neonatal mouse brains and total proteins were extractedfrom whole harvested brains. A GFP-specific antibody was used to detectthe GFP expression in each brain sample and a standard GFP protein wasused for quantification. N=5 animals per condition. Error bars representthe mean±SEM

FIG. 38 Amino acid sequence of the VP1 capsid protein of AAV3B. Thehighlighted residues represent the residues that are identical to theones in AAV-tt at corresponding positions. The internal serine residueat position 312 is underlined.

FIG. 39 Amino acid sequence of the VP1 capsid protein of AAV-LK03.

LIST OF SEQUENCES SEQ ID NO:1 is the amino acid sequence of wild-typeadeno-associated virus 2 capsid protein VP1 (see FIG. 1).

SEQ ID NO:2 is the amino acid sequence of true-type adeno-associatedvirus 2 (ttAAV2) capsid protein (see FIG. 2).

SEQ ID NO:3 is the amino acid sequence of wild-type adeno-associatedvirus 1 capsid protein VP1 (see FIG. 3).

SEQ ID NO:4 is the amino acid sequence of wild-type adeno-associatedvirus 5 capsid protein VP1 (see FIG. 4).

SEQ ID NO:5 is the amino acid sequence of wild-type adeno-associatedvirus 6 capsid protein VP1 (see FIG. 5).

SEQ ID NO:6 is the amino acid sequence of wild-type adeno-associatedvirus 8 capsid protein VP1 (see FIG. 6).

SEQ ID NO:7 is the amino acid sequence of wild-type adeno-associatedvirus 9 capsid protein VP1 (see FIG. 7).

SEQ ID NO:8 is the amino acid sequence of wild-type adeno-associatedvirus 10 Upenn capsid protein VP1 (see FIG. 8).

SEQ ID NO:9 is the amino acid sequence of wild-type adeno-associatedvirus 10 japanese capsid protein VP1 (see FIG. 9).

SEQ ID NO:10 is the consensus amino acid sequence for adeno-associatedviruses shown in FIG. 9.

SEQ ID NO:11 is the amino acid sequence of wild-type adeno-associatedvirus 3B capsid protein VP1 (see FIG. 38).

SEQ ID NO:12 is the amino acid sequence of adeno-associated virus LK-03capsid protein VP1 (see FIG. 39).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to a recombinantadeno-associated virus (AAV) vector. The rAAV vector typically comprisesa variant capsid protein which differs compared to a wild-type AAVcapsid protein. The variant capsid protein may advantageously conferenhanced infectivity of the vector in brain and/or eye, making thevector particularly suited to delivery of therapeutic agents by genetherapy into these tissues.

RECOMBINANT AAV VECTOR

The present disclosure provides a recombinant adeno-associated virus(rAAV) vector. “AAV” is an abbreviation for adeno-associated virus, andmay be used to refer to the virus itself or derivatives thereof. Theterm covers all subtypes and both naturally occurring and recombinantforms, except where required otherwise. The abbreviation “rAAV” refersto recombinant adeno-associated virus, also referred to as a recombinantAAV vector (or “rAAV vector”). The term “AAV” includes, for example, AAVtype 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4(AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAVtype 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10, includingAAVrh10), AAV type 12 (AAV-12), avian AAV, bovine AAV, canine AAV,equine AAV, primate AAV, non-primate AAV, and ovine AAV. “Primate AAV”refers to AAV that infect primates, “non-primate AAV” refers to AAV thatinfect non-primate mammals, “bovine AAV” refers to AAV that infectbovine mammals, and so on.

The genomic sequences of various serotypes of AAV, as well as thesequences of the native terminal repeats (TRs), Rep proteins, and capsidsubunits are known in the art. Such sequences may be found in theliterature or in public databases such as GenBank. See, e.g., GenBankAccession Numbers NC-002077 (AAV-1), AF063497 (AAV-1), NC-001401(AAV-2), AF043303 (AAV-2), NC-001729 (AAV-3), NC-001829 (AAV- 4), U89790(AAV-4), NC-006152 (AAV-5), AF513851 (AAV-7), AF513852 (AAV-8), andNC-006261 (AAV-8); the disclosures of which are incorporated byreference herein. See also, e.g., Srivistava et al. (1983) J. Virology45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al.(1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996)Virology 221:208; Shade et al.,(1986) J. Virol. 58:921; Gao et al.(2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology33:375-383; international patent publications WO 00/28061, WO 99/61601,WO 98/11244; and U.S. Pat. No. 6,156,303.

An “rAAV vector” as used herein refers to an AAV vector comprising apolynucleotide sequence not of AAV origin (i.e., a polynucleotideheterologous to AAV), typically a sequence of interest for the genetictransformation of a cell. In some embodiments, the heterologouspolynucleotide may be flanked by at least one, and sometimesby two, AAVinverted terminal repeat sequences (ITRs). The term rAAV vectorencompasses both rAAV vector particles and rAAV vector plasmids. An rAAVvector may either be single-stranded (ssAAV) or self-complementary(scAAV).

An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refersto a viral particle composed of at least one AAV capsid protein(typically by all of the capsid proteins of a wild-type AAV) and anencapsidated polynucleotide rAAV vector. If the particle comprises aheterologous polynucleotide (i.e. a polynucleotide other than awild-type AAV genome such as a transgene to be delivered to a mammaliancell), it is typically referred to as an “rAAV vector particle” orsimply an “rAAV vector”. Thus, production of rAAV particle necessarilyincludes production of rAAV vector, as such a vector is contained withinan rAAV particle.

“Recombinant,” as used herein means that the vector, polynucleotide,polypeptide or cell is the product of various combinations of cloning,restriction or ligation steps (e.g. relating to a polynucleotide orpolypeptide comprised therein), and/or other procedures that result in aconstruct that is distinct from a product found in nature. A recombinantvirus or vector is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

VARIANT AAV CAPSID PROTEINS

The rAAV vectors described herein comprise a variant AAV capsid protein.By “variant” it is meant that the AAV capsid protein differs from acorresponding wild type AAV capsid protein of the same serotype. Forinstance, the variant AAV capsid protein may comprise one or more aminoacid substitutions with respect to the corresponding wild type AAVcapsid protein. In this context, “corresponding” refers to a capsidprotein of the same serotype, i.e. a variant AAV1 capsid proteincomprises one or more amino acid substitutions with respect to thecorresponding wild type AAV1 capsid protein, a variant AAV2 capsidprotein comprises one or more amino acid substitutions with respect tothe corresponding wild type AAV2 capsid protein, and so on.

The variant AAV capsid protein may comprise, for example, 1 to 50, 1 to30, 1 to 20 or 1 to 15 amino acid substitutions with respect to the wildtype AAV capsid protein. Preferably the variant AAV capsid proteincomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acidsubstitutions with respect to the corresponding wild type AAV capsidprotein. In preferred embodiments, the variant AAV capsid proteinretains at least 70%, at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% sequence identity to thewild type capsid protein.

In embodiments of the present invention, the variant AAV capsid proteincomprises at least one amino acid substitution with respect to a wildtype AAV capsid protein at a position corresponding to one or more ofthe following positions in an AAV2 capsid protein sequence: 125, 151,162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593. Inthis context, “corresponding” refers to a position in any AAV capsidprotein sequence (e.g. in an AAV2 protein sequence or a non-AAV2 capsidprotein sequence) which corresponds to one of the above positions inAAV2 capsid protein. In one embodiment, the at least one amino acidsubstitution is present at one or more of the following positions in anAAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499,533, 546, 548, 585, 588 and/or 593; or at one or more correspondingpositions in an alternative AAV capsid protein sequence.

In general, AAV capsid proteins include VP1, VP2 and VP3. In a preferredembodiment, the capsid protein comprises AAV capsid protein VP1.

NUCLEIC ACID AND AMINO ACID SEQUENCES AND SEQUENCE IDENTITY

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs, and may beinterrupted by non-nucleotide components. If present, modifications tothe nucleotide structure may be imparted before or after assembly of thepolymer. The term polynucleotide, as used herein, refers interchangeablyto double- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of the invention described herein that is apolynucleotide encompasses both the double-stranded form and each of twocomplementary single-stranded forms known or predicted to make up thedouble-stranded form.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The terms also encompass an amino acid polymer that has beenmodified; for example, disulfide bond formation, glycosylation,lipidation, phosphorylation, or conjugation with a labeling component.Polypeptides such as anti-angiogenic polypeptides, neuroprotectivepolypeptides, and the like, when discussed in the context of deliveringa gene product to a mammalian subject, and compositions therefor, referto the respective intact polypeptide, or any fragment or geneticallyengineered derivative thereof, which retains the desired biochemicalfunction of the intact protein. Similarly, references to nucleic acidsencoding anti-angiogenic polypeptides, nucleic acids encodingneuroprotective polypeptides, and other such nucleic acids for use indelivery of a gene product to a mammalian subject (which may be referredto as “transgenes” to be delivered to a recipient cell), includepolynucleotides encoding the intact polypeptide or any fragment orgenetically engineered derivative possessing the desired biochemicalfunction.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same whencomparing the two sequences. Sequence similarity can be determined in anumber of different manners. To determine sequence identity, sequencescan be aligned using the methods and computer programs, including BLAST,available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Anotheralignment algorithm is FASTA, available in the Genetics Computing Group(GCG) package, from Madison, Wis., USA, a wholly owned subsidiary ofOxford Molecular Group, Inc. Other techniques for alignment aredescribed in Methods in Enzymology, vol. 266: Computer Methods forMacromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press,Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Ofparticular interest are alignment programs that permit gaps in thesequence. The Smith-Waterman is one type of algorithm that permits gapsin sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also,the GAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970)

Of interest is the BestFit program using the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981) todetermine sequence identity. The gap generation penalty will generallyrange from 1 to 5, usually 2 to 4 and in many embodiments will be 3. Thegap extension penalty will generally range from about 0.01 to 0.20 andin many instances will be 0.10. The program has default parametersdetermined by the sequences inputted to be compared. Preferably, thesequence identity is determined using the default parameters determinedby the program. This program is available also from Genetics ComputingGroup (GCG) package, from Madison, Wis., USA.

Another program of interest is the FastDB algorithm. FastDB is describedin Current Methods in Sequence Comparison and Analysis, MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp.127-149, 1988, Alan R. Liss, Inc. Percent sequence identity iscalculated by FastDB based upon the following parameters:

Mismatch Penalty: 1.00;

Gap Penalty: 1.00;

Gap Size Penalty: 0.33; and

Joining Penalty: 30.0.

VARIANT AAV2 CAPSID PROTEIN

In one embodiment, the vector comprises a variant AAV2 capsid protein.In this embodiment, the variant AAV2 capsid protein comprises at leastone amino acid substitution at one or more of the following positions inan AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499,533, 546, 548, 585, 588 and/or 593.

The sequence of wild type AAV2 capsid protein VP1 is known, and is shownin FIG. 1 (SEQ ID NO:1). Wild type AAV2 capsid protein sequences arealso available from database accession no.s: NC-001401; UniProt P03135;NCBI Reference Sequence: YP_680426.1; GenBank: AAC03780.1.

Preferably the variant AAV2 capsid protein has at least 70%, at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity to SEQ ID NO:1. In a preferredembodiment, the variant AAV2 capsid protein comprises a sequence of SEQID NO:2, or a sequence having at least 70%, at least 80%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity thereto.

In one embodiment, the variant AAV2 capsid protein comprises one or moreof the following residues: I125, A151, S162, S205, S312, M457, A492,D499, Y533, D546, G548, S585, T588 and/or S593. In a preferredembodiment, the variant AAV2 capsid protein comprises one or more of thefollowing amino acid substitutions with respect to a wild type AAV2capsid protein: V125I, V151A, A162S, T205S, N312S, Q457M, S492A, E499D,F533Y, G546D, E548G, R585S, R588T and/or A593S.

COMBINATIONS OF MUTATIONS IN AAV2 CAPSID PROTEIN

The variant AAV2 capsid protein may comprise any combination of theabove amino acid substitutions. Therefore in particular embodiments, thevariant AAV2 capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 amino acid substitutions selected from the list above. Inone embodiment, the variant AAV2 capsid protein comprises all 14 aminoacid substitutions disclosed above, e.g. the variant AAV2 capsid proteincomprises a sequence of SEQ ID NO:2 (i.e. ttAAV2 or AAV2-TT as referredto herein).

In further embodiments, the variant AAV2 capsid protein may comprise asub-set of the above 14 mutations. Without being bound by theory, inindividual embodiments, the variant AAV2 capsid protein may comprise thefollowing residues, which are divided below into functional groups:

1) S585 and/or T588; these residues may be associated with decreasedheparin binding and increased spread of the virus in heparin sulphateproteoglycan-rich brain tissue;

2) S312; this internal serine residue may play a role in capsid-DNAinteractions;

3) D546 and/or G548; these residues may be involved in interactions withneutralizing antibodies and thus contribute to in vivo transductioncharacteristics;

4) S593; this residue is located in the groove betweenthreefold-proximal spikes;

5) M457, A492, D499 and/or Y533; these four amino acids may be involvedin receptor binding and are closely situated on the threefold spikes;

6) I125, A151, S162 and/or S205; these residues may be associated withPLA2 activity and/or trafficking of the incoming virus.

It will be appreciated that also contemplated herein are correspondingsub-groups comprising mutations corresponding to the above residues whenpresent at corresponding positions in further AAV serotypes (see below).

In preferred embodiments, the variant AAV2 capsid protein comprises fouror more mutations at the positions mentioned above which may beassociated with receptor binding, i.e. residues 457, 492, 499 and 533.Thus it is particularly preferred that the variant AAV2 capsid proteincomprises the following residues M457, A492, D499 and Y533.

In some preferred embodiments, the variant AAV2 capsid protein is notmutated with respect to the wild type AAV2 capsid protein at position312, e.g. the variant AAV2 capsid protein comprises the residue N312(which is present in the wild type AAV2 capsid protein). Thus in someembodiments, the variant AAV2 capsid protein may comprise 1 to 13 of thespecific mutations mentioned above, but not the mutation N312S.

VARIANT AAV CAPSID PROTEINS FROM OTHER SEROTYPES

In further embodiments, the variant AAV capsid protein is from analternative AAV serotype, i.e. an AAV serotype other than AAV2. Forinstance, the variant AAV capsid protein may be derived from an AAV1,AAV3B, AAV-LK03, AAV5, AAV6, AAV8, AAV9 or AAV10 (e.g. AAVrh10) capsidprotein.

In these embodiments, the variant AAV capsid protein comprises at leastone amino acid substitution at one or more positions corresponding tothose described above with respect to AAV2. In other words, the variantAAV capsid protein comprises at least one amino acid substitution at aposition in an alternative (i.e. non-AAV2) AAV capsid protein sequencewhich corresponds to positions 125, 151, 162, 205, 312, 457, 492, 499,533, 546, 548, 585, 588 and/or 593 in an AAV2 capsid protein sequence.

Those skilled in the art would know, based on a comparison of the aminoacid sequences of capsid proteins of various AAV serotypes, how toidentify positions in capsid proteins from alternative AAV serotypeswhich correspond to positions 125, 151, 162, 205, 312, 457, 492, 499,533, 546, 548, 585, 588 and/or 593 in an AAV2 capsid protein. Inparticular, such positions can easily be identified by sequencealignments as known in the art and described herein. For instance, onesuch sequence alignment is provided in FIG. 9.

Of particular relevance in this context are positions in alternative AAVcapsid protein sequences which correspond in three-dimensional space topositions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585,588 and/or 593 in an AAV2 capsid protein. Methods for three-dimensionalmodelling and alignment of protein structures are well known in the art,and can be used to identify such corresponding positions in non-AAV2capsid protein sequences. Exemplary 3D alignments of AAV2 capsid proteinsequences with capsid protein sequences of alternative AAV serotypes(e.g. AAV1, AAV5, AAV6, AAV8 and AAV9) are shown in FIGS. 21 to 25 anddiscussed below. A skilled person can perform similar 3D alignments withcapsid proteins from further serotypes, e.g. AAV2, AAV3, AAV7, AAV10 andAAV12), and identify positions in such sequences which correspond withto the positions defined above in AAV2.

VARIANT AAV1 CAPSID PROTEIN

In one embodiment, the vector comprises a variant AAV1 capsid protein.In this embodiment, the variant AAV1 capsid protein comprises at leastone amino acid substitution at one or more of the following positions inthe AAV1 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493,500, 534, 547, 549, 586, 589 and/or 594. These positions in AAV1 capsidprotein VP1 correspond to those disclosed above in relation to AAV2.

The sequence of wild type AAV1 capsid protein VP1 is known, and is shownin FIG. 3 (SEQ ID NO:3). A wild type AAV1 capsid protein sequences isalso available from database accession no.: NC-002077. Preferably thevariant AAV1 capsid protein has at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to SEQ ID NO:3.

Wild type AAV1 capsid protein VP1 already contains the followingresidues at positions which correspond to amino acid residues which arepresent in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2,ttAAV2), but not wild type AAV2 (SEQ ID NO:1): S205 (aligns with S205 inttAAV2); G549 (aligns with G548 in ttAAV2); S586 (aligns with S585 inttAAV2); and T589 (aligns with T588 in ttAAV2). Accordingly, in apreferred embodiment, the variant AAV1 capsid protein comprises one ormore of the following amino acid substitutions with respect to a wildtype AAV1 capsid protein: V125I, Q151A, T162S, N313S, N458M, K493A,N500D, F534Y, S547D, and/or G594S. Typically such a variant AAV1 capsidprotein may share one or more functional properties with the variantAAV2 capsid protein (SEQ ID NO:2, ttAAV2), e.g. may confer increasedinfectivity and/or transduction of neuronal of retinal tissue comparedto wild type AAV1 capsid protein.

In alternative embodiments, the variant AAV1 capsid protein comprisesone or more amino acid substitutions which correspond to reversions ofmutations present in ttAAV2 back to the wild type AAV2 sequence. Forinstance, the variant AAV1 capsid protein may comprise one or more ofthe following substitutions: S205T, G549E, S586R and/or T589R. Typicallysuch a variant AAV1 capsid protein may share one or more functionalproperties with the wild type AAV2 capsid protein (SEQ ID NO:1), e.g.may confer reduced infectivity and/or transduction of neuronal ofretinal tissue compared to wild type AAV1 capsid protein.

VARIANT AAV5 CAPSID PROTEIN

In one embodiment, the vector comprises a variant AAV5 capsid protein.In this embodiment, the variant AAV5 capsid protein comprises at leastone amino acid substitution at one or more of the following positions inthe AAV5 capsid protein sequence: 124, 150, 153, 195, 303, 444, 479,486, 520, 533, 537, 575, 578 and/or 583. These positions in AAV5 capsidprotein VP1 correspond to those disclosed above in relation to AAV2.

The sequence of wild type AAV5 capsid protein VP1 is known, and is shownin FIG. 4 (SEQ ID NO:4). A wild type AAV5 capsid protein sequences isalso available from database accession no.: AF085716. Preferably thevariant AAV5 capsid protein has at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to SEQ ID NO:4.

Wild type AAV5 capsid protein VP1 already contains the followingresidues at positions which correspond to amino acid residues which arepresent in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2,ttAAV2), but not wild type AAV2 (SEQ ID NO:1): G537 (aligns with G548 inttAAV2); S575 (aligns with S585 in ttAAV2); T578 (aligns with T588 inttAAV2). Accordingly, in a preferred embodiment, the variant AAV5 capsidprotein comprises one or more of the following amino acid substitutionswith respect to a wild type AAV5 capsid protein: V124I, K150A, K1535,A195S, R3035, T444M, S479A, V486D, T520Y, P533D, and/or G583S. Typicallysuch a variant AAV5 capsid protein may share one or more functionalproperties with the variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2),e.g. may confer increased infectivity and/or transduction of neuronal ofretinal tissue compared to wild type AAV5 capsid protein.

In alternative embodiments, the variant AAV5 capsid protein comprisesone or more amino acid substitutions which correspond to reversions ofmutations present in ttAAV2 back to the wild type AAV2 sequence. Forinstance, the variant AAV5 capsid protein may comprise one or more ofthe following substitutions: G537E, S575R and/or T578R. Typically such avariant AAV5 capsid protein may share one or more functional propertieswith the wild type AAV2 capsid protein (SEQ ID NO:1), e.g. may conferreduced infectivity and/or transduction of neuronal of retinal tissuecompared to wild type AAV5 capsid protein.

VARIANT AAV6 CAPSID PROTEIN

In one embodiment, the vector comprises a variant AAV6 capsid protein.In this embodiment, the variant AAV6 capsid protein comprises at leastone amino acid substitution at one or more of the following positions inthe AAV6 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493,500, 534, 547, 549, 586, 589 and/or 594. These positions in AAV6 capsidprotein VP1 correspond to those disclosed above in relation to AAV2.

The sequence of wild type AAV6 capsid protein VP1 is known, and is shownin FIG. 5 (SEQ ID NO:5). A wild type AAV6 capsid protein sequences isalso available from database accession no.: AF028704. Preferably thevariant AAV6 capsid protein has at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to SEQ ID NO:5.

Wild type AAV6 capsid protein VP1 already contains the followingresidues at positions which correspond to amino acid residues which arepresent in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2,ttAAV2), but not wild type AAV2 (SEQ ID NO:1): S205 (aligns with S205 inttAAV2); G549 (aligns with G548 in ttAAV2); S586 (aligns with S585 inttAAV2); T589 (aligns with T588 in ttAAV2). Accordingly, in a preferredembodiment, the variant AAV6 capsid protein comprises one or more of thefollowing amino acid substitutions with respect to a wild type AAV6capsid protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y,S547D, and/or G594S. Typically such a variant AAV6 capsid protein mayshare one or more functional properties with the variant AAV2 capsidprotein (SEQ ID NO:2, ttAAV2), e.g. may confer increased infectivityand/or transduction of neuronal of retinal tissue compared to wild typeAAV6 capsid protein.

In alternative embodiments, the variant AAV6 capsid protein comprisesone or more amino acid substitutions which correspond to reversions ofmutations present in ttAAV2 back to the wild type AAV2 sequence. Forinstance, the variant AAV6 capsid protein may comprise one or more ofthe following substitutions: S205T, G549E, S586R and/or T589R. Typicallysuch a variant AAV6 capsid protein may share one or more functionalproperties with the wild type AAV2 capsid protein (SEQ ID NO:1), e.g.may confer reduced infectivity and/or transduction of neuronal ofretinal tissue compared to wild type AAV6 capsid protein.

VARIANT AAV8 CAPSID PROTEIN

In one embodiment, the vector comprises a variant AAV8 capsid protein.In this embodiment, the variant AAV8 capsid protein comprises at leastone amino acid substitution at one or more of the following positions inthe AAV8 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495,502, 536, 549, 551, 588, 591 and/or 596. These positions in AAV8 capsidprotein VP1 correspond to those disclosed above in relation to AAV2.

The sequence of wild type AAV8 capsid protein VP1 is known, and is shownin FIG. 6 (SEQ ID NO:6). A wild type AAV8 capsid protein sequences isalso available from database accession no.: NC_006261. Preferably thevariant AAV8 capsid protein has at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to SEQ ID NO:6.

Wild type AAV8 capsid protein VP1 already contains the followingresidues at positions which correspond to amino acid residues which arepresent in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2,ttAAV2), but not wild type AAV2 (SEQ ID NO:1): S315 (aligns with S312 inttAAV2); T591 (aligns with T588 in ttAAV2). Accordingly, in a preferredembodiment, the variant AAV8 capsid protein comprises one or more of thefollowing amino acid substitutions with respect to a wild type AAV8capsid protein: V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y,N549D, A551G, Q588S and/or G596S. Typically such a variant AAV8 capsidprotein may share one or more functional properties with the variantAAV2 capsid protein (SEQ ID NO:2, ttAAV2), e.g. may confer increasedinfectivity and/or transduction of neuronal of retinal tissue comparedto wild type AAV8 capsid protein.

In alternative embodiments, the variant AAV8 capsid protein comprisesone or more amino acid substitutions which correspond to reversions ofmutations present in ttAAV2 back to the wild type AAV2 sequence. Forinstance, the variant AAV8 capsid protein may comprise one or more ofthe following substitutions: S315N and/or T591R. Typically such avariant AAV8 capsid protein may share one or more functional propertieswith the wild type AAV2 capsid protein (SEQ ID NO:1), e.g. may conferreduced infectivity and/or transduction of neuronal of retinal tissuecompared to wild type AAV8 capsid protein.

In one embodiment, the variant AAV8 capsid protein comprises an aminoacid substitution with respect to a wild type AAV8 capsid protein atposition 315 in an AAV8 capsid protein sequence. For instance, thevariant AAV8 capsid protein may comprise the residue N315. Thus in oneembodiment the variant AAV8 capsid protein comprises the amino acidsubstitution S315N with respect to a wild type AAV8 capsid protein.

VARIANT AAV9 CAPSID PROTEIN

In one embodiment, the vector comprises a variant AAV9 capsid protein.In this embodiment, the variant AAV9 capsid protein comprises at leastone amino acid substitution at one or more of the following positions inthe AAV9 capsid protein sequence: 125, 151, 162, 205, 314, 458, 493,500, 534, 547, 549, 586, 589 and/or 594. These positions in AAV9 capsidprotein VP1 correspond to those disclosed above in relation to AAV2.

The sequence of wild type AAV9 capsid protein VP1 is known, and is shownin FIG. 7 (SEQ ID NO:7). A wild type AAV9 capsid protein sequences isalso available from database accession no.: AY530579. Preferably thevariant AAV9 capsid protein has at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to SEQ ID NO:7.

Wild type AAV9 capsid protein VP1 already contains the followingresidues at positions which correspond to amino acid residues which arepresent in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2,ttAAV2), but not wild type AAV2 (SEQ ID NO:1): S162 (aligns with S162 inttAAV2); S205 (aligns with S205 in ttAAV2); G549 (aligns with G548 inttAAV2); S586 (aligns with S585 in ttAAV2). Accordingly, in a preferredembodiment, the variant AAV9 capsid protein comprises one or more of thefollowing amino acid substitutions with respect to a wild type AAV9capsid protein: L125S, Q151A, N314S, Q458M, V493A, E500D, F534Y, G547D,A589T and/or G594S. Typically such a variant AAV9 capsid protein mayshare one or more functional properties with the variant AAV2 capsidprotein (SEQ ID NO:2, ttAAV2), e.g. may confer increased infectivityand/or transduction of neuronal of retinal tissue compared to wild typeAAV9 capsid protein.

In alternative embodiments, the variant AAV9 capsid protein comprisesone or more amino acid substitutions which correspond to reversions ofmutations present in ttAAV2 back to the wild type AAV2 sequence. Forinstance, the variant AAV9 capsid protein may comprise one or more ofthe following substitutions: S162A, S205T, G549E and/or S586R. Typicallysuch a variant AAV9 capsid protein may share one or more functionalproperties with the wild type AAV2 capsid protein (SEQ ID NO:1), e.g.may confer reduced infectivity and/or transduction of neuronal ofretinal tissue compared to wild type AAV9 capsid protein.

VARIANT AAV10 CAPSID PROTEIN

In one embodiment, the vector comprises a variant AAV10 capsid protein.As used herein, “AAV10” includes AAVrh10. In this embodiment, thevariant AAV10 (e.g. AAVrh10) capsid protein comprises at least one aminoacid substitution at one or more of the following positions in the AAV10capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536,549, 551, 588, 591 and/or 596. These positions in AAV10 capsid proteinVP1 correspond to those disclosed above in relation to AAV2.

The sequence of wild type AAV10 capsid protein VP1 is known, and isshown in FIG. 8 (SEQ ID NO:8). Preferably the variant AAV10 capsidprotein has at least 70%, at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto SEQ ID NO:8.

Wild type AAV10 capsid protein VP1 already contains the followingresidue at a position which corresponds to an amino acid residue whichis present in the variant AAV2 capsid protein disclosed above (SEQ IDNO:2, ttAAV2), but not wild type AAV2 (SEQ ID NO:1): G551 (aligns withG548 in ttAAV2). Accordingly, in a preferred embodiment, the variantAAV10 capsid protein comprises one or more of the following amino acidsubstitutions with respect to a wild type AAV10 capsid protein: V125I,Q151A, K163S, A206S, N315S, T460M, L495A, N502D, F536Y, G549D, Q588S,A591T and/or G596S. Typically such a variant AAV10 capsid protein mayshare one or more functional properties with the variant AAV2 capsidprotein (SEQ ID NO:2, ttAAV2), e.g. may confer increased infectivityand/or transduction of neuronal of retinal tissue compared to wild typeAAV10 capsid protein.

In alternative embodiments, the variant AAV10 capsid protein comprisesan amino acid substitution which corresponds to a reversion of amutations present in ttAAV2 back to the wild type AAV2 sequence. Forinstance, the variant AAV10 capsid protein may comprise the followingsubstitution: G551E. Typically such a variant AAV10 capsid protein mayshare one or more functional properties with the wild type AAV2 capsidprotein (SEQ ID NO:1), e.g. may confer reduced infectivity and/ortransduction of neuronal of retinal tissue compared to wild type AAV10capsid protein.

VARIANT AAV3B CAPSID PROTEIN

In one embodiment, the vector comprises a variant AAV3B capsid protein.In this embodiment, the variant AAV3B capsid protein may comprise anamino acid substitution with respect to a wild type AAV3B capsid proteinat position 312. For instance, the variant AAV3B capsid protein maycomprise the residue N312. Thus in one embodiment the variant AAV8capsid protein comprises the amino acid substitution S312N with respectto a wild type AAV8 capsid protein. In further embodiments, the variantAAV3B capsid protein may comprise one or more additional mutations atpositions which correspond to those disclosed above in relation to AAV2.

The sequence of wild type AAV3B capsid protein VP1 is known, and isshown in FIG. 38 (SEQ ID NO:11). A wild type AAV3B capsid proteinsequence is also available from NCBI database accession no. AF028705.Preferably the variant AAV3B capsid protein has at least 70%, at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity to SEQ ID NO:11.

VARIANT AAV-LK03 CAPSID PROTEIN

In one embodiment, the vector comprises a variant AAV-LK03 capsidprotein. In this embodiment, the variant AAV-LK03 capsid protein maycomprise an amino acid substitution at position 312 with respect to aAAV-LK03 capsid protein sequence as defined in SEQ ID NO:12. Forinstance, the variant AAV-LK03 capsid protein may comprise the residueN312. Thus in one embodiment the variant AAV-LK03 capsid proteincomprises the amino acid substitution S312N with respect to a AAV-LK03capsid protein sequence as defined in SEQ ID NO:12. In furtherembodiments, the variant AAV-LK03 capsid protein may comprise one ormore additional mutations at positions which correspond to thosedisclosed above in relation to AAV2.

The sequence of wild type AAV-LK03 capsid protein VP1 is known, and isshown in FIG. 39 (SEQ ID NO:12). A AAV-LK03 capsid protein sequence isalso disclosed in WO 2013/029030 as sequence number 31 therein.Preferably the variant AAV-LK03 capsid protein has at least 70%, atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity to SEQ ID NO:12.

GENE PRODUCTS

In one embodiment, the rAAV further comprises a heterologous nucleicacid comprising a nucleotide sequence encoding a gene product. A “gene”refers to a polynucleotide containing at least one open reading framethat is capable of encoding a particular protein after being transcribedand translated. A “gene product” is a molecule resulting from expressionof a particular gene. Gene products include, e.g., a polypeptide, anaptamer, an interfering RNA, an mRNA, and the like.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is being compared. Forexample, a polynucleotide introduced by genetic engineering techniquesinto a plasmid or vector derived from a different species is aheterologous polynucleotide. A promoter removed from its native codingsequence and operatively linked to a coding sequence with which it isnot naturally found linked is a heterologous promoter. Thus, forexample, an rAAV that includes a heterologous nucleic acid encoding aheterologous gene product is an rAAV that includes a nucleic acid notnormally included in a naturally-occurring, wild-type AAV, and theencoded heterologous gene product is a gene product not normally encodedby a naturally-occurring, wild-type AAV.

In some embodiments, the gene product is an interfering RNA. In someembodiments, the gene product is an aptamer. In some embodiments, thegene product is a polypeptide.

Interfering RNA

Where the gene product is an interfering RNA (RNAi), suitable RNAiinclude RNAi that decrease the level of an apoptotic or angiogenicfactor in a cell. For example, an RNAi can be an shRNA or siRNA thatreduces the level of a gene product that induces or promotes apoptosisin a cell. Genes whose gene products induce or promote apoptosis arereferred to herein as “pro-apoptotic genes” and the products of thosegenes (mRNA; protein) are referred to as “pro-apoptotic gene products.”Pro-apoptotic gene products include, e.g., Bax, Bid, Bak, and Bad geneproducts. See, e.g., U.S. Pat. No. 7,846,730.

Interfering RNAs could also be against an angiogenic product, forexample VEGF (e.g., Cand5; see, e.g., U.S. Patent Publication No.2011/0143400; U.S. Patent Publication No. 2008/0188437; and Reich et al.(2003) Mol. Vis. 9:210), VEGFR1 (e.g., Sirna-027; see, e.g., Kaiser etal. (2010) Am. J. Ophthalmol. 150:33; and Shen et al. (2006) Gene Ther.13:225), or VEGFR2 (Kou et al. (2005) Biochem. 44: 15064). See also,U.S. Pat. Nos. 6,649,596, 6,399,586, 5,661,135, 5,639,872, and5,639,736; and U.S. Pat. Nos. 7,947,659 and 7,919,473.

A “small interfering” or “short interfering RNA” or siRNA is a RNAduplex of nucleotides that is targeted to a gene interest (a “targetgene”). An “RNA duplex” refers to the structure formed by thecomplementary pairing between two regions of a RNA molecule. siRNA is“targeted” to a gene in that the nucleotide sequence of the duplexportion of the siRNA is complementary to a nucleotide sequence of thetargeted gene. In some embodiments, the length of the duplex of siRNAsis less than 30 nucleotides. In some embodiments, the duplex can be 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11or 10 nucleotides in length. In some embodiments, the length of theduplex is 19-25 nucleotides in length. The RNA duplex portion of thesiRNA can be part of a hairpin structure. In addition to the duplexportion, the hairpin structure may contain a loop portion positionedbetween the two sequences that form the duplex. The loop can vary inlength. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13nucleotides in length. The hairpin structure can also contain 3′ or 5′overhang portions. In some embodiments, the overhang is a 3′ or a 5′overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.

A “short hairpin RNA,” or shRNA, is a polynucleotide construct that canbe made to express an interfering RNA such as siRNA.

Aptamers

Where the gene product is an aptamer, exemplary aptamers of interestinclude an aptamer against vascular endothelial growth factor (VEGF).See, e.g., Ng et al. (2006) Nat. Rev. Drug Discovery 5: 123; and Lee etal. (2005) Proc. Natl. Acad. Sci. USA 102: 18902. Also suitable for useis a PDGF-specific aptamer, e.g., E10030; see, e.g., Ni and Hui (2009)Ophthalmologica 223:401; and Akiyama et al. (2006) J. Cell Physiol.207:407).

Polypeptides

In one embodiment, the gene product is a therapeutic protein. A“therapeutic” peptide or protein is a peptide or protein that mayalleviate or reduce symptoms that result from an absence or defect in aprotein in a cell or subject. Alternatively, a “therapeutic” peptide orprotein is one that otherwise confers a benefit to a subject, e.g.,anti-degenerative effects.

Where the gene product is a polypeptide, the polypeptide is generally apolypeptide that enhances function of a cell, for example a cell presentin neuronal, retinal or liver tissue, e.g., a hepatocyte, a neuron, aglial cell, a rod or cone photoreceptor cell, a retinal ganglion cell, aMuller cell, a bipolar cell, an amacrine cell, a horizontal cell, or aretinal pigmented epithelial cell.

Exemplary polypeptides include neuroprotective polypeptides (e.g., GDNF,CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides (e.g., a solublevascular endothelial growth factor (VEGF) receptor; a VEGF-bindingantibody; a VEGF-binding antibody fragment (e.g., a single chainanti-VEGF antibody); endostatin; tumstatin; angiostatin; a soluble Fitpolypeptide (Lai et al. (2005) Mol. Ther. 12:659); an Fc fusion proteincomprising a soluble Fit polypeptide (see, e.g., Pechan et al. (2009)Gene Ther. 16: 10); pigment epithelium-derived factor (PEDF); a solubleTie-2 receptor; etc.); tissue inhibitor of metalloproteinases-3(TIMP-3); a light-responsive opsin, e.g., a rhodopsin; anti- apoptoticpolypeptides (e.g., Bcl-2, Bcl-X1); and the like. Suitable polypeptidesinclude, but are not limited to, glial derived neurotrophic factor(GDNF); fibroblast growth factor 2; neurturin (NTN); ciliaryneurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4(NT4); brain derived neurotrophic factor (BDNF); epidermal growthfactor; rhodopsin; X-linked inhibitor of apoptosis; and Sonic hedgehog.Suitable polypeptides are disclosed, for example, in WO WO 2012/145601.

Exemplary polypeptides for gene deliver to the liver include, forexample, PBGD (porphobilinogen deaminase) IDUA (iduronidase) Fah(fumarylacetoacetate hydrolyase) A1AT (alpha(1)-antitrypsin),1A1(hUGT1A1) (uridine disphoshate glucuronyltransferase), HCCS1(hepatocellular carcinoma suppressor 1), CD (cytosine deaminase), SOCS3(suppressor of cytokine signaling 3), TNF (tumor necrosis factor),thymidine kinase, IL-24 (interleukin-24), IL-12 (interleukin-12), andTRAIL (tumor necrosis factor-related apoptosis-inducing ligand).

Regulatory Sequences

In some embodiments, a nucleotide sequence encoding a gene product ofinterest is operably linked to a constitutive promoter. In otherembodiments, a nucleotide sequence encoding a gene product of interestis operably linked to an inducible promoter. In some instances, anucleotide sequence encoding a gene product of interest is operablylinked to a tissue specific or cell type specific regulatory element.

For example, in some instances, a nucleotide sequence encoding a geneproduct of interest is operably linked to a hepatocyte-specific,neuron-specific or photoreceptor-specific regulatory element (e.g., aphotoreceptor-specific promoter), e.g., a regulatory element thatconfers selective expression of the operably linked gene in a neuron orphotoreceptor cell. Suitable photoreceptor-specific regulatory elementsinclude, e.g., a rhodopsin promoter; a rhodopsin kinase promoter (Younget al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterasegene promoter (Nicoud et al. (2007) J. Gene Med. 9: 1015); a retinitispigmentosa gene promoter (Nicoud et al. (2007) supra); aninterphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoudet al. (2007) supra); an IRBP gene promoter (Yokoyama et al. (1992) ExpEye Res. 55:225). Suitable neuronal-specific promoters includeneuron-specific enolase (NSE) promoter, Andersen et al. Cell. Mol.Neurobiol., 13:503-15 (1993; neurofilament light-chain gene promoter,Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:561 1-5 (1991); and theneuron-specific vgf gene promoter, Piccioli et al., Neuron, 15:373-84(1995)]; among others. Suitable hepatocyte-specific promoters include analbumin promoter (Heard et al., Mol Cell Biol 1987; 7: 2425) or an alpha1-antitrypsin promoter (Hafenrichter et al. Blood 1994; 84, 3394-404).

A “control element” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules that contributes to thefunctional regulation of a polynucleotide, including replication,duplication, transcription, splicing, translation, or degradation of thepolynucleotide. The regulation may affect the frequency, speed, orspecificity of the process, and may be enhancing or inhibitory innature. Control elements known in the art include, for example,transcriptional regulatory sequences such as promoters and enhancers. Apromoter is a DNA region capable under certain conditions of binding RNApolymerase and initiating transcription of a coding region usuallylocated downstream (in the 3′ direction) from the promoter.

“Operatively linked” or “operably linked” refers to a juxtaposition ofgenetic elements, wherein the elements are in a relationship permittingthem to operate in the expected manner. For instance, a promoter isoperatively linked to a coding region if the promoter helps initiatetranscription of the coding sequence. There may be intervening residuesbetween the promoter and coding region so long as this functionalrelationship is maintained.

The term “promoters” or “promoter” as used herein can refer to a DNAsequence that is located adjacent to a DNA sequence that encodes arecombinant product. A promoter is preferably linked operatively to anadjacent DNA sequence. A promoter typically increases an amount ofrecombinant product expressed from a DNA sequence as compared to anamount of the expressed recombinant product when no promoter exists. Apromoter from one organism can be utilized to enhance recombinantproduct expression from a DNA sequence that originates from anotherorganism. For example, a vertebrate promoter may be used for theexpression of jellyfish GFP in vertebrates. In addition, one promoterelement can increase an amount of recombinant products expressed formultiple DNA sequences attached in tandem. Hence, one promoter elementcan enhance the expression of one or more recombinant products. Multiplepromoter elements are well-known to persons of ordinary skill in theart.

The term “enhancers” or “enhancer” as used herein can refer to a DNAsequence that is located adjacent to the DNA sequence that encodes arecombinant product. Enhancer elements are typically located upstream ofa promoter element or can be located downstream of or within a codingDNA sequence (e.g., a DNA sequence transcribed or translated into arecombinant product or products). Hence, an enhancer element can belocated 100 base pairs, 200 base pairs, or 300 or more base pairsupstream or downstream of a DNA sequence that encodes recombinantproduct. Enhancer elements can increase an amount of recombinant productexpressed from a DNA sequence above increased expression afforded by apromoter element. Multiple enhancer elements are readily available topersons of ordinary skill in the art.

PHARMACEUTICAL COMPOSITIONS

The present disclosure provides a pharmaceutical composition comprising:a) a rAAV vector, as described above; and b) a pharmaceuticallyacceptable carrier, diluent, excipient, or buffer. In some embodiments,the pharmaceutically acceptable carrier, diluent, excipient, or bufferis suitable for use in a human.

Such excipients, carriers, diluents, and buffers include anypharmaceutical agent that can be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to,liquids such as water, saline, glycerol and ethanol.

Pharmaceutically acceptable salts can be included therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles. A widevariety of pharmaceutically acceptable excipients are known in the artand need not be discussed in detail herein. Pharmaceutically acceptableexcipients have been amply described in a variety of publications,including, for example, A. Gennaro (2000) “Remington: The Science andPractice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins;Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Anselet al., eds., 7(th) ed., Lippincott, Williams, & Wilkins; and Handbookof Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3 rd ed.Amer. Pharmaceutical Assoc.

In particular embodiments, the present invention provides apharmaceutical composition comprising a rAAV vector as described abovein a pharmaceutically-acceptable carrier or other medicinal agents,pharmaceutical agents, carriers, adjuvants, diluents, etc. Forinjection, the carrier will typically be a liquid. For other methods ofadministration, the carrier may be either solid or liquid, such assterile, pyrogen-free water or sterile pyrogen-free phosphate-bufferedsaline solution. For inhalation administration, the carrier will berespirable, and will preferably be in solid or liquid particulate form.As an injection medium, it is preferred to use water that contains theadditives usual for injection solutions, such as stabilizing agents,salts or saline, and/or buffers.

By “pharmaceutically acceptable” it is meant a material that is notbiologically or otherwise undesirable, e.g., the material may beadministered to a subject without causing any undesirable biologicaleffects. Thus, such a pharmaceutical composition may be used, forexample, in transfection of a cell ex vivo or in administering a viralparticle or cell directly to a subject.

METHODS OF DELIVERING A GENE PRODUCT TO A TISSUE OR CELL (FOR EXAMPLE AHEPATIC, NEURONAL OR RETINAL TISSUE OR CELL) AND TREATMENT METHODS

The methods of the present invention provide a means for deliveringheterologous nucleic acid sequences into a host tissue or cell,including both dividing and non-dividing cells. The vectors and otherreagents, methods and pharmaceutical formulations of the presentinvention are additionally useful in a method of administering a proteinor peptide to a subject in need thereof, as a method of treatment orotherwise. In this manner, the protein or peptide may thus be producedin vivo in the subject. The subject may be in need of the protein orpeptide because the subject has a deficiency of the protein or peptide,or because the production of the protein or peptide in the subject mayimpart some therapeutic effect, as a method of treatment or otherwise,and as explained further below.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease or at risk of acquiring the diseasebut has not yet been diagnosed as having it; (b) inhibiting the disease,i.e., arresting its development; and (c) relieving the disease, i.e.,causing regression of the disease.

In general, the present invention may be employed to deliver any foreignnucleic acid with a biological effect to treat or ameliorate thesymptoms associated with any disorder related to gene expression in anyorgan, tissue or cell, especially those associated with e.g. the liver,brain or eye. Illustrative disease states include, but are not limitedto: lysosomal storage disease, acute intermittent porphyria, ornithinetranscarbamylase deficiency, alpha(1)-antitrypsin deficiency, acuteliver failure, Pompe disease, Tyrosinemia, Crigler-Najjar syndrome,hepatitis, cirrhosis, hepatocellular carcinoma, AIDS, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, epilepsy, and other neurological disorders, cancer (e.g.brain cancer), retinal degenerative diseases and other diseases of theeye.

Gene transfer has substantial potential use in understanding andproviding therapy for disease states. There are a number of inheriteddiseases in which defective genes are known and have been cloned. Insome cases, the function of these cloned genes is known. In general, theabove disease states fall into two classes: deficiency states, usuallyof enzymes, which are generally inherited in a recessive manner, andunbalanced states, at least sometimes involving regulatory or structuralproteins, which are inherited in a dominant manner. For deficiency statediseases, gene transfer could be used to bring a normal gene intoaffected tissues for replacement therapy, as well as to create animalmodels for the disease using antisense mutations. For unbalanced diseasestates, gene transfer could be used to create a disease state in a modelsystem, which could then be used in efforts to counteract the diseasestate. Thus the methods of the present invention permit the treatment ofgenetic diseases. As used herein, a disease state is treated bypartially or wholly remedying the deficiency or imbalance that causesthe disease or makes it more severe. The use of site-specificintegration of nucleic sequences to cause mutations or to correctdefects is also possible.

In one aspect the present invention provides a method of delivering agene product to a tissue or cell (e.g. a hepatic, neuronal or retinaltissue or cell) in a subject, the method comprising administering to thesubject a rAAV vector as described above. The gene product can be apolypeptide or an interfering RNA (e.g., an shRNA, an siRNA, and thelike), or an aptamer, e.g. as described above. The cell may, forexample, be a blood cell, stem cell, bone marrow (e.g. hematopoietic)cell, liver cell, cancer cell, vascular cell, pancreatic cell, neuralcell, glial cell, ocular or retinal cell, epithelial or endothelialcell, dendritic cell, fibroblast, lung cell, muscle cell, cardiac cell,intestinal cell or renal cell. Similarly the tissue may, for example, beselected from blood, bone marrow, muscle tissue (e.g. skeletal muscle,cardiac muscle or smooth muscle including vascular smooth muscle),central or peripheral nervous system tissue (e.g. brain, neuronal tissueor retinal tissue), pancreatic tissue, liver tissue, kidney tissue, lungtissue, intestinal tissue or heart tissue.

Delivering a gene product to a retinal cell can provide for treatment ofa retinal disease. The retinal cell can be a photoreceptor, a retinalganglion cell, a Muller cell, a bipolar cell, an amacrine cell, ahorizontal cell, or a retinal pigmented epithelial cell. In some cases,the retinal cell is a photoreceptor cell, e.g., a rod or cone cell.Similarly, delivering a gene product to a neuronal tissue or cell canprovide for treatment of a neurological disorder. The gene product maybe delivered to various cell types present in neuronal tissue, e.g.neurons or glial cells (e.g. astrocytes, oligodendrocytes and so on).Delivering a gene product to the liver may provide treatment for ahepatic disorder. The gene product may be delivered to, for example,hepatocytes.

The present disclosure provides a method of treating a disease (e.g. ahepatic, neurological or ocular disease), the method comprisingadministering to an individual in need thereof an effective amount of arAAV vector as described above. A subject rAAV vector can beadministered via intracranial injection, intracerebral injection,intraocular injection, by intravitreal injection, retinal injection,sub-retinal injection, intravenous injection or by any other convenientmode or route of administration.

Further exemplary modes of administration include oral, rectal,transmucosal, topical, transdermal, inhalation, parenteral (e.g.,intravenous, subcutaneous, intradermal, intramuscular, andintraarticular) administration, and the like, as well as direct tissueor organ injection, alternatively, intrathecal, direct intramuscular,intraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspensions in liquid prior to injection, or asemulsions. Alternatively, one may administer the virus in a local ratherthan systemic manner, for example in a depot or sustained-releaseformation.

Recombinant virus vectors are preferably administered to the subject inan amount that is sufficient to result in infection (or transduction)and expression of the heterologous nucleic acid sequence in cells (e.g.liver, neuronal or retinal cells) of the subject. Preferably the targetcells are hepatocytes, neural cells (including cells of the central andperipheral nervous systems, in particular, brain cells) or retinalcells. In some cases, the retinal cell is a photoreceptor cell (e.g.,rods and/or cones). In other cases, the retinal cell is an RGC cell. Inother cases, the retinal cell is an RPE cell. In other cases, retinalcells may include amacrine cells, bipolar cells, and horizontal cells.

Preferably the vector is administered in a therapeutically effectiveamount. A “therapeutically-effective” amount as used herein is an amountof that is sufficient to alleviate (e.g., mitigate, decrease, reduce) atleast one of the symptoms associated with a disease state. Alternativelystated, a “therapeutically-effective” amount is an amount that issufficient to provide some improvement in the condition of the subject.A “therapeutically effective amount” will fall in a relatively broadrange that can be determined through experimentation and/or clinicaltrials. For example, for in vivo injection, a therapeutically effectivedose will be on the order of from about 10⁶ to about 10¹⁵ of rAAVvirions, e.g., from about 10⁸ to 10¹² rAAV virions. For in vitrotransduction, an effective amount of rAAV virions to be delivered tocells will be on the order of from about 10⁸ to about 10¹³ of the rAAVvirions. Other effective dosages can be readily established by one ofordinary skill in the art through routine trials establishing doseresponse curves.

In some embodiments, more than one administration (e.g., two, three,four or more administrations) may be employed to achieve the desiredlevel of gene expression over a period of various intervals, e.g.,daily, weekly, monthly, yearly, etc.

Neurological diseases which may be treated include any diseaseassociated with the brain or CNS, including psychiatric diseases.Diseases of the brain fall into two general categories: (a) pathologicprocesses such as infections, trauma and neoplasm; and (b) diseasesunique to the nervous system which include diseases of myelin anddegeneration of neurons. Disease from either category may be treated.For example, the neurological disease may be selected fromneurodegenerative diseases such as Alzheimer's Disease, Parkinson'sDisease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophyand cerebella degeneration; schizophrenia; epilepsy; ischemia-relateddisease and stroke; demyelinating diseases such as multiple sclerosis,perivenous encephalitis, leukodystrophies such as metachromaticleukodystrophy due to deficiency of arylsulfatase A, Krabbe's diseasedue to deficiency of galactocerebroside beta-galactosidase,adrenoleukodystrophy and adrenomyeloneuropathy; post-viral diseases suchas progressive multifocal leukoencephalopathy, acute disseminatedencephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis;mitochondrial encephalomyopathies; neurological cancers, such as primarybrain tumors including glioma, meningioma, neurinoma, pituitary adenoma,medulloblastoma, craniopharyngioma, hemangioma, epidermoid, sarcoma andintracranial metastasis from other tumor sources; neurologicalinfections or neurological inflammatory conditions.

Ocular diseases that can be treated using a subject method include, butare not limited to, acute macular neuroretinopathy; Behcet's disease;choroidal neovascularization; diabetic uveitis; histoplasmosis; maculardegeneration, such as acute macular degeneration, non-exudative agerelated macular degeneration and exudative age related maculardegeneration; edema, such as macular edema, cystoid macular edema anddiabetic macular edema; multifocal choroiditis; ocular trauma whichaffects a posterior ocular site or location; ocular tumors; retinaldisorders, such as central retinal vein occlusion, diabetic retinopathy(including proliferative diabetic retinopathy), proliferativevitreoretinopathy (PVR), retinal arterial occlusive disease, retinaldetachment, uveitic retinal disease; sympathetic opthalmia; VogtKoyanagi-Harada (VKH) syndrome; uveal diffusion; a posterior ocularcondition caused by or influenced by an ocular laser treatment;posterior ocular conditions caused by or influenced by a photodynamictherapy; photocoagulation, radiation retinopathy; epiretinal membranedisorders; branch retinal vein occlusion; anterior ischemic opticneuropathy; non-retinopathy diabetic retinal dysfunction; retinoschisis;retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy;Stargardt disease (fundus flavimaculatus); inherited maculardegeneration; chorioretinal degeneration; Leber congenital amaurosis;congenital stationary night blindness; choroideremia; Bardet-Biedlsyndrome; macular telangiectasia; Leber's hereditary optic neuropathy;retinopathy of prematurity; and disorders of color vision, includingachromatopsia, protanopia, deuteranopia, and tritanopia.

Diseases of the liver which may be treated include, for example,lysosomal storage diseases, e.g. acute intermittent porphyria, ornithinetranscarbamylase deficiency, Wilson's disease, mucopolysaccharidoses(e.g. MPS type I or MPS type VI), Sly syndrome, Pompe disease,tyrosinemia, alpha(1)-antitrypsin deficiency, Crigler-Najjar syndrome;hepatitis A, B or C; liver cirrhosis; liver cancer, e.g. hepatocellularcarcinoma; or acute liver failure.

The present invention finds use in both veterinary and medicalapplications. Suitable subjects include both avians and mammals, withmammals being preferred. The term “avian” as used herein includes, butis not limited to, chickens, ducks, geese, quail, turkeys and pheasants.The term “mammal” as used herein includes, but is not limited to,humans, bovines, ovines, caprines, equines, felines, canines,lagomorphs, etc. Human subjects are the most preferred. Human subjectsinclude fetal, neonatal, infant, juvenile and adult subjects.

TRANSDUCTION OF TISSUE (FOR EXAMPLE HEPATIC, NEURONAL OR RETINAL TISSUE)

In some embodiments, the rAAV vectors disclosed herein exhibit increasedtransduction of a tissue (e.g. hepatic, neuronal and/or retinaltissues), e.g. compared to a corresponding AAV vector (from the sameserotype) comprising a wild type AAV capsid protein. For example, therAAV vector may exhibit at least 10%, 50%, 100%, 500% or 1000% increasedinfectivity, compared to the infectivity by an AAV virion comprising thecorresponding wild type AAV capsid protein.

In further embodiments, the rAAV vectors disclosed herein mayselectively or specifically infect a tissue (e.g. hepatic, neuronal orretinal tissues), e.g. show increased transduction of hepatic, neuronalor retinal cells compared to other cell types. For instance, the rAAVvector may exhibit at least 10%, 50%, 100%, 500% or 1000% increasedinfectivity of a particular cell type (e.g. hepatic, neuronal or retinalcells), compared to another cell type (e.g. non-hepatic, non-neuronaland/or non-retinal cells). For instance, the rAAV vector may selectivelyinfect hepatocytes, neurons and/or photoreceptor cells compared to cellsoutside the liver, brain and/or eye.

Where the recombinant AAV vector exhibits increased transduction of aneuronal or retinal tissue, e.g. where the vector is used to treat aneurological or ocular disorder, the vector preferably comprises avariant AAV2 capsid protein.

Where the recombinant AAV vector exhibits increased transduction ofliver tissue, e.g. where the vector is used to treat a hepatic disorder,the vector preferably comprises a variant AAV3B, AAV-LK03 or AAV8 capsidprotein.

NUCLEIC ACIDS AND HOST CELLS

The present disclosure provides an isolated nucleic acid comprising anucleotide sequence that encodes a variant adeno-associated virus (AAV)capsid protein as described above. The isolated nucleic acid can becomprised in an AAV vector, e.g., a recombinant AAV vector.

A recombinant AAV vector comprising such a variant AAV capsidprotein-encoding sequence can be used to generate a recombinant AAVvirion (i.e. a recombinant AAV vector particle). Thus, the presentdisclosure provides a recombinant AAV vector that, when introduced intoa suitable cell, can provide for production of a recombinant AAV virion.

The present invention further provides host cells, e.g., isolated(genetically modified) host cells, comprising a subject nucleic acid. Asubject host cell can be an isolated cell, e.g., a cell in in vitroculture. A subject host cell is useful for producing a subject rAAVvirion, as described below. Where a subject host cell is used to producea subject rAAV virion, it is referred to as a “packaging cell.” In someembodiments, a subject host cell is stably genetically modified with asubject nucleic acid. In other embodiments, a subject host cell istransiently genetically modified with a subject nucleic acid.

A subject nucleic acid is introduced stably or transiently into a hostcell, using established techniques, including, but not limited to,electroporation, calcium phosphate precipitation, liposome-mediatedtransfection, and the like. For stable transformation, a subject nucleicacid will generally further include a selectable marker, e.g., any ofseveral well-known selectable markers such as neomycin resistance, andthe like.

A subject host cell is generated by introducing a subject nucleic acidinto any of a variety of cells, e.g., mammalian cells, including, e.g.,murine cells, and primate cells (e.g., human cells). Suitable mammaliancells include, but are not limited to, primary cells and cell lines,where suitable cell lines include, but are not limited to, 293 cells,COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2fibroblasts, CHO cells, and the like. Non-limiting examples of suitablehost cells include, e.g., HeLa cells (e.g., American Type CultureCollection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61,CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells(e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No.CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No.CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonickidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. Asubject host cell can also be made using a baculovirus to infect insectcells such as Sf9 cells, which produce AAV (see, e.g., U.S. Pat. No.7,271,002; U.S. patent application Ser. No. 12/297,958).

In some embodiments, a subject genetically modified host cell includes,in addition to a nucleic acid comprising a nucleotide sequence encodinga variant AAV capsid protein, as described above, a nucleic acid thatcomprises a nucleotide sequence encoding one or more AAV rep proteins.In other embodiments, a subject host cell further comprises an rAAVvector. An rAAV virion can be generated using a subject host cell.Methods of generating an rAAV virion are described in, e.g., U.S. PatentPublication No. 2005/0053922 and U.S. Patent Publication No.2009/0202490.

As used herein, “packaging” refers to a series of intracellular eventsthat result in the assembly and encapsidation of an AAV particle. AAV“rep” and “cap” genes refer to polynucleotide sequences encodingreplication and encapsidation proteins of adeno-associated virus. AAVrep and cap are referred to herein as AAV “packaging genes.” Assemblyassociated protein (AAP) is the product of an open reading frame withinthe cap gene, and may also be required for packaging.

A “helper virus” for AAV refers to a virus that allows AAV (e.g.wild-type AAV) to be replicated and packaged by a mammalian cell. Avariety of such helper viruses for AAV are known in the art, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC. Viruses of the herpesfamily include, for example, herpes simplex viruses (HSV) andEpstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) andpseudorabies viruses (PRV); which are also available from depositoriessuch as ATCC.

“Helper virus function(s)” refers to function(s) encoded in a helpervirus genome which allow AAV replication and packaging (in conjunctionwith other requirements for replication and packaging described herein).As described herein, “helper virus function” may be provided in a numberof ways, including by providing helper virus or providing, for example,polynucleotide sequences encoding the requisite function(s) to aproducer cell in trans. For example, a plasmid or other expressionvector comprising nucleotide sequences encoding one or more adenoviralproteins is transfected into a producer cell along with an rAAV vector.

An “infectious” virus or viral particle is one that comprises acompetently assembled viral capsid and is capable of delivering apolynucleotide component into a cell for which the viral species istropic. The term does not necessarily imply any replication capacity ofthe virus. Assays for counting infectious viral particles are describedelsewhere in this disclosure and in the art. Viral infectivity can beexpressed as the ratio of infectious viral particles to total viralparticles. Methods of determining the ratio of infectious viral particleto total viral particle are known in the art. See, e.g., Grainger et al.(2005) Mol. Ther. 11:S337 (describing a TCID50 infectious titer assay);and Zolotukhin et al. (1999) Gene Ther. 6:973.

A “replication-competent” virus (e.g. a replication-competent AAV)refers to a phenotypically wild-type virus that is infectious, and isalso capable of being replicated in an infected cell (i.e. in thepresence of a helper virus or helper virus functions). In the case ofAAV, replication competence generally requires the presence offunctional AAV packaging genes. In general, rAAV vectors as describedherein are replication-incompetent in mammalian cells (especially inhuman cells) by virtue of the lack of one or more AAV packaging genes.Typically, such rAAV vectors lack any AAV packaging gene sequences inorder to minimize the possibility that replication competent AAV aregenerated by recombination between AAV packaging genes and an incomingrAAV vector. In many embodiments, rAAV vector preparations as describedherein are those which contain few if any replication competent AAV(rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10²rAAV particles, less than about 1 rcAAV per 10⁴ rAAV particles, lessthan about 1 rcAAV per 10⁸ rAAV particles, less than about 1 rcAAV per10¹² rAAV particles, or no rcAAV).

An “isolated” nucleic acid, vector, virus, virion, host cell, or othersubstance refers to a preparation of the substance devoid of at leastsome of the other components that may also be present where thesubstance or a similar substance naturally occurs or is initiallyprepared from. Thus, for example, an isolated substance may be preparedby using a purification technique to enrich it from a source mixture.Enrichment can be measured on an absolute basis, such as weight pervolume of solution, or it can be measured in relation to a second,potentially interfering substance present in the source mixture.Increasing enrichments of the embodiments of this disclosure areincreasingly more isolated. An isolated nucleic acid, vector, virus,host cell, or other substance is in some embodiments purified, e.g.,from about 80% to about 90% pure, at least about 90% pure, at leastabout 95% pure, at least about 98% pure, or at least about 99%, or more,pure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The following example is provided to illustrate certain embodiments ofthe invention. It is not intended to limit the invention in any way.

EXAMPLES Example 1

In this Example, the inventors designed and constructed a novel AAV2vector designated ttAAV2 (as in true-type). In addition, the novelvector was tested in a number of animal models (rats, mice and neonatalmice) in order to evaluate whether ttAAV2 behaved differently ascompared to the tissue culture adapted (wild type) AAV2. The inventorsdemonstrated that ttAAV2 has advantages for gene delivery over AAV2, andis particularly useful for in vivo transduction of brain or eye tissueswith heterologous sequences.

Methods

1. Cloning: The capsid gene of wtAAV2 was taken from our producerplasmid pDG (FIG. 10). This plasmid contains wtAAV2 rep and cap genes.Subfragments of the capsid gene (pDG nucleotides A:3257-3759,B:4025-4555, C:4797-5287 and D:5149-5425, respectively) were subclonedinto pBS for subsequent mutagenesis. Four mutations were introduced intofragment A via site-directed mutagenesis resulting in a construct thatencodes for amino acid (AA) changes V125I, V151A, A162S and T205S.Fragment B was mutated to encode the single AA change, N312S. Fragment Cwas mutated to encode the AA exchanges Q457M, S492A, E499D, F533Y,G546D, E548G, R585S, R588T, and A593S. Upon confirmation of successfulmutagenesis fragments A-C were re-cloned into pDG, resulting in aproducer plasmid (pDG-ttAAV2) that would support the production of arecombinant virus that is encapsidated by ttAAV2 capsid.

2. ttAAV2-GFP Viral Vector Production Purification and Titration.

Vector production was established following the standard protocolsemploying co-transfection of rAAV plasmids with pDG, which provides boththe Ad helper functions as well as the AAV rep and cap genes. A varietyof rAAV plasmids were used to generate recombinant plasmids.

pTR-UF11 (CAG-GFP) was used as the rAAV plasmid. 8×10⁸ 293 cells wereseeded per cell factory (CF10). 14-18 hours later, the cells weretransfected with pDG or pDG-rrAAV2 and prAAV (e.g. pTR-UF11) using theCaPO₄ co-precipitation method. After 72 hours the cells were harvestedand resuspended in lysis buffer (20 mMTris-HCl, pH8, 150 mM NaCl, 0.5%deoxycholate). The cell pellets were lysed by four cycles of freeze andthaw to release the virus, where each cycle consists of 30 minutes at−80° C. followed by 30 minutes at 37° C. After the last thaw the lysatewas treated with benzonase at a concentration of 50 U/ml and incubatedfor 30 min at 37° C. The recombinant virus was purified using gravityflow columns.

Purification. As a first step, the crude lysate was clarified bycentrifugation at 4000 g for 15 minutes and applied to the pre-formediodixanol step gradient. The viral fraction was then collected andre-buffered into Lactate Ringer's solution as well as concentrated usingAmicon centrifugation filters.

Subsequently, purity of the viral preparations were assessed by SDSpolyacrylamide electrophoresis and titered using real time PCR methods.The crude extract contained 4.5×10¹² particles; the collected viralfractions contained 1.5×10¹² particles. At this point the purificationmethod recovered ca. 33% of the virus present in the crude extract.

3. rAAV Vector Production and Purification (Alternative Method)

In an alternative embodiment to that at point 2 above, the rAAV2 vectoris produced as follows. To produce rAAV2 virions, 5×10⁸ 293T cells wereseeded per cell factory (CF10). 14-18 hours later, the cells were doubletransfected with the GFP-containing vector PD10-pST2-CMV-GFP, and eitherthe pDG or the pDG capsid mutant (pDG-ttAAV2) to produce AAV2-CMV-GFPwild-type or true-type vectors, respectively. The double transfectionswere realised using PEI-max from Polysciences at a ratio of 3.5 ml ofPEI per mg of DNA. The cells were harvested after 72 hours of incubationat 37° C. by centrifugating the media and cells at 2200 rpm for 10minutes at 4° C. The supernatant was removed and kept for furthertreatment, and the cells pellets were resuspended in lysis buffer (0.15M NaCl, 50 mM Tris-HCl [pH 8.8]).

The cell pellets were then lysed by 4 cycles of freeze and thaw torelease the virus, where each cycle consists of 30 minutes at −80° C.followed by 30 minutes at 37° C. After the last thaw the lysate wastreated with benzonase at a concentration of 150 U/ml and incubated at37° for 30 minutes. The lysate was then spun at 2000 rpm for 20 minutesto clarify the lysate. The supernatant was filtered using a 0.22 μmcellulose acetate filter and the recombinant AAV2 virus preparationswere purified by FPLC using the AKTApurifier chromatography system (GEHealthcare) and an AVB sepharose affinity column (bfr. A: PBS, pH 8; bfrB: 0.5M glycine, pH2.7). The collected fractions were dialysed againstPBS overnight and the viral preparations were then titered by SDSpolyacrylamide electrophoresis and real time PCR methods.

Results In Vivo Transduction and Spread of ttAAV2

The ttAAV2 vector was tested in vivo in order to assess the bioactivityof the modified virus in such a context. Samples of AAV2-CMV-GFP WT andTT viruses were prepared for injections into a number of in vivo models.For this purpose we concentrated the viruses, as only limited volumes ofvectors can be injected in vivo. We then performed a qPCR and SDS-PAGEto assess the new titres of the concentrated vectors (FIG. 11).

After qPCR and protein gel analysis we obtained the following newtitres: AAV2-CMV-GFP TT at 1.33×10¹² viral genomes/ml and AAV2-CMV-GFPWT at 1.25×10¹² viral genomes/ml.

The capsid titres were as follow: AAV2-CMV-GFP TT at 8.89×10¹²capsids/ml and AAV2-CMV-GFP WT at 7.83×10¹² capsids/ml. The titresdiffer between the genome copies and the capsid copies as the SDS-PAGEalso shows empty capsids, which are normally generated duringrecombinant AAV vectors production, hence the capsid titre is higherthan the viral genome titre obtained from the qPCR.

Transduction in Rat Brain

The rAAV2 TT and WT viruses were injected in the substantia nigra or inthe striatum of wild-type rats, with 3 rats being injected percondition, at a dose of 2×10⁹ vg or 3.5×10⁹ vg per injection. After 28days brains were dissected and tissue sections were prepared forimmunofluorescence analysis. The primary data are shown in FIG. 12 andFIG. 26.

Both the rAAV2 TT and WT viruses were able to transduce neuronal andglial cells from each injection site, albeit with varying efficiencies.By comparison, we observed that the TT vector transduced brain tissuesmore efficiently and spread more from the site of injection than the WTvector. Furthermore, we observed the presence of transduced neurons inthe parafascicularis nucleus, an area of the hypothalamus, afterstriatal injection of the rAAV2 TT. This indicates that the TT vectorwas able to travel from the transduced cell bodies at the site ofinjection to the hypothalamus by active transport along the neuronprojections, highlighting a strong ability for retrograde transport.This retrograde transport ability has been lost in the tissue-cultureadapted WT rAAV2 vector as no transduced cells could be observed in thesame area (see FIG. 26).

Taken together, in rat brains these results indicate a significantlyincreased spread and transduction efficiency by ttAAV2 as compared to atitre-matched wtAAV2. Furthermore, AAV2 TT displays evidence for verygood retrograde transport ability, which has been lost in the AAV2 WTvirus.

Transduction in a Mouse Eye Model

ttAAV2 and wt-AAV2 from the same batch as was used for our rat brainstudies was injected into adult mouse eyes at a dose of 2×10⁹ vg pereye. To avoid animal to animal variability, each mouse received aninjection of rAAV2 TT in one eye and an injection of rAAV2 WT in thecontra-lateral eye. Three different routes of intra-ocular injectionswere analysed: intra-cameral, intra-vitreal and sub-retinal. The animalswere harvested and GFP expression was assessed by immunofluorescenceafter 6 weeks. The results are shown in FIG. 13. Together, these dataindicate a marked enhancement of transduction of photoreceptor cells byttAAV2 following sub-retinal injection, in terms of both level andnumbers of photoreceptor cells transduced, if compared to wtAAV2 (whichwas used in the successful RPE65 clinical trial).

Transduction in Neonatal Mouse Model

In summary, both ttAAV2 and wtAAV2 GFP vectors were injected into mouseneonates. Two routes of injections were tested, intra-venous injectionand intra-cranial injections. After 4 weeks, the animals were sacrificedand all tissues were harvested from all mice. We have analysed thebrain, which after harvesting was visualised by direct fluorescence ofthe organ on a fluorescence microscope. The results are shown in FIG.14. The results of intracranial and systemic injections are discussed inmore detail below.

Intracranial Injections

5×10¹⁰ vg of either vector were injected in the lateral ventricle of P1neonates. The animals were sacrificed 4 weeks post-injection and thebrains were dissected, sectioned and stained using an anti-GFP antibody.The results are shown in FIG. 27.

As observed in adult rat brains, these data indicate that AAV2 TTdisplays enhanced transduction of mouse brain tissues and higher spreadafter intracranial injection as compared to the AAV2 WT vector. Whenobserving the stained sections at a higher magnification, thedifferences in transduction efficiency between both vectors were furtherhighlighted: the TT vector performed better both in terms of level ofexpression and of number of cells transduced. AAV2 TT and WT seem tohave the same cell type affinity, each displaying transduction ofneuronal as well as glial cells, suggesting that the differencesobserved are differences in efficiency rather than in cell-typespecificity (FIG. 28).Taken together these data indicate that ttAAV2shows much enhanced transduction of mouse brain tissues after i.c.injection as compared to wtAAV2-based vectors. In addition, someevidence is suggestive for transduction of the ependymal cell layerlining the ventricles when ttAAV2 vectors are used. This phenomenon isnot visible with the wtAAV2 vector.

Systemic Injections

Intrajugular injections of 2×10¹¹ vg of either vector were done in P1neonates. The animals were sacrificed 4 weeks post-injection and variousorgans were harvested and assessed for GFP transduction byimmunohistochemistry using an anti-GFP antibody (brain, liver, heart,muscle, lungs, spleen and kidney). Results of the brains staining areshown in FIG. 29 and high magnification pictures are presented in FIG.30.

We observed good transgene expression in the CNS after systemicinjection of AAV2 TT. The AAV2 WT vector performed worse in comparison,with only few transduced neurons observed.

In order to assess the overall biodistribution of the AAV2 TT vector weassessed the level of transduction obtained in various tissues aftersystemic injection. The harvested organs were fixed, paraffin embedded,sectioned and stained for GFP expression (FIG. 31).

These data indicate that the AAV-TT vector doesn't seem to have a strongaffinity for other organs but instead displays specificity mainly forneuronal tissues. This observation could prove beneficial for thetreatment of neuronal genetic disorders by intravenous injections of AAVas it ensures that the vector will not transduce non-target peripheralorgans but mainly only the brain via this injection route.

Together, our in vivo data suggests that ttAAV2 has extraordinarytransduction characteristics in eye and brain tissues, displayingspecificity for neuronal tissues almost exclusively.

Example 2 Additional Considerations

Without being bound by theory, it is believed that the mutations presentin ttAAV2 compared to wtAAV2 comprise the following functional groups:

1) heparin binding residues located on the AAV2 capsid threefold spikes(S585 and T588); it is believed that these residues are responsible forheparin binding of the wtAAV2 capsid. In ttAAV2 these are replaced andwe assume that this replacement supports the spread of the virus inheparan sulphate proteoglycan-rich brain tissue.

2) the single amino acid change in ttAAV2 that is located on theinternal side of the capsid (S312); this internal serine residue mightplay a role in capsid-DNA interactions, thereby potentially contributingto, either virus stability, genome packaging or genome release duringinfection.

3) two spatially close amino acids (D546 and G548) located in the groovebetween the threefold-proximal spikes on AAV capsid structure; theseresidues might be involved in interactions with neutralizing antibodiesand thus contribute to in vivo transduction characteristics.

4) a single isolated amino acid change (S593) located in the groovebetween threefold-proximal spikes;

5) four amino acids believed to be involved in receptor binding andclosely situated on the threefold spikes (M457, A492, D499 and Y533);

6) the four remaining amino acid changes situated in VP1/VP2 primarysequence (I125, A151, S162 and S205); these residues are within thecapsid region that is not part of VP3. It is known that VP1/VP2 specificregions within the virus capsid contain PLA2 activity which might beinvolved in trafficking of the incoming virus. Changes in this regionhave been shown to influence viral infectivity.

The three-dimensional positions of these mutations in the AAV2 capsidprotein are known and are shown in FIGS. 15 to 20. Correspondingpositions can be identified in the AAV capsid proteins from otherserotypes (see below). To further characterize the ttAAV2 and the roleof individual amino acid changes that are responsible for the improvedttAAV2 phenotype, the ttAAV2 capsid can be mutated in order to reverseindividual chosen amino acids (or groups of amino acids, e.g. based ongroups 1 to 6 discussed above) to their corresponding sequence in thewild-type AAV2 capsid. Each mutant vector can then be analysed usingmethods as described in the examples above.

For instance the various mutant vectors, expressing GFP, can besubmitted to a first screen in an animal model by intracranial (IC)injection in CD1 neonatal mice. The GFP signal obtained in the injectedbrains from each mutant vector is then observed by fluorescencemicroscopy and compared to that obtained from the originalGFP-expressing wtAAV2 and ttAAV2 vectors. Upon identification of a newphenotype (i.e. the abolition of the ttAAV2-specific strong GFPexpression when mutating a particular amino acid group), the injectedbrains are then further sectioned and analysed by immunohistochemistry.

In parallel, these selected mutant vectors are submitted to a secondscreen by adult rat intracranial injections. Additionally, relevantmutant combinations are analysed by intravenous (IV) injections intoneonatal mice in order to evaluate the biodistribution of the vectors.Selected organs (heart, lung, liver, spleen, kidney, muscle) are thenprocessed for immunohistochemistry and evaluation of GFP expression.

Analysis of the Contribution of Each TT-Specific Residue to theEfficiency and Biodistribution of the Vector

To further characterize the AAV2 TT and pinpoint the amino acid changesthat are responsible for the improved TT phenotype, the AAV2 TT capsidwas mutated step by step in order to reverse the chosen amino acids totheir corresponding sequence in the wild-type AAV2 capsid. This strategyenabled us to discern more specifically the contribution of each of the14 amino acid mutations towards the phenotype of AAV2 TT and to define aminimal true-type vector, containing only the necessary mutations.

As discussed above, we grouped the 14 TT-specific residues into groupsbased on their positions on the AAV capsid and their associatedpotential contributions to the transduction profile. The various AAV-TTmutants were screened by intracranial injections in neonatal mousebrains or in adult rat brains in order to observe whether the reversionof some TT-specific residues to the WT equivalents was associated with aloss of phenotype, thereby identifying the important amino acid changesamongst the 14 TT residues.

The Heparin Binding Site (HBS)

It has been shown that residues 585 and 588 are responsible for heparinbinding of the AAV2 WT capsid. In AAV-TT these are replaced and weassumed that this replacement support the spread of the virus in heparinsulphate proteoglycan-rich brain tissue. These two residues are situatedon the three-fold proximal spikes of the AAV2 capsid structure (see FIG.16).

We abolished the AAV2 WT heparin binding ability by engineering thechanges R585S and R588T on the WT capsid (AAV2-HBnull), i.e. mutatingthe residues to the true-type equivalents. This first analysis wasperformed in order to ascertain that AAV-TT was not merely an AAV2without heparin binding site, able to spread more, but that some of theother 12 amino acid changes also play a role in the improved AAV-TTtransduction profile.

Intracranial Injections in Adult Rat

Titer-matched AAV2-TT, AAV2-WT and AAV2-HBnull vectors were injected inthe substantia nigra or in the striatum of wild-type rats at a dose of3.5×10⁹ vg per injection. After 28 days brains were dissected and tissuesections were prepared for immunohistochemistry analysis of GFPexpression (FIG. 32).

We observed a strong GFP expression in the thalamus and in thesubstantia nigra after striatum injection of AAV-TT virus, highlightingthe strong retrograde transport ability of the vector. In comparison,AAV2-HBnull and AAV2 WT displayed much less—if any—retrograde transportthan AAV-TT as very few cell bodies were transduced in these area afterstriatal injections. This observation showed that AAV2-HBnull isdifferent to AAV-TT and that the absence of heparin binding ability onthe AAV2 capsid contributes to the good spread of the true-type vectorin the brain. However it is not sufficient to explain its improvedtransduction profile.

The Residues S312, D546-G548 and S593

The S312 residue is the only TT-specific change that is located on theinternal side of the AAV2 capsid. Our assumption was that this internalresidue might play a role in capsid-DNA interactions, therebypotentially contributing to either virus stability, genome packaging orgenome release during infection.

The D and G residues at positions 546 and 548, respectively, are locatedin the groove between the proximal 3-fold peaks.

A single isolated serine, S593, is situated in the groove betweenthreefold-proximal spikes.

The positions of these TT-specific residues on the three-dimensionalstructure of the AAV2 capsid are presented in FIGS. 17 to 19. Giventheir less prominent position in the structure we hypothesised thatthese residues might not contribute to the ttAAV2 transductionphenotype.

Neonatal Mice Intracranial Injections

The mutation S312N was engineered on the AAV2-TT capsid to create theTT-S312N mutant. The mutations D546G and G548E were engineered on theAAV2-TT capsid to generate the AAV-TT-DG mutant. The mutation S593A wasengineered on the AAV2-TT capsid to create the AAV-TT-S593A mutant.

By reverting these chosen TT amino acids to their corresponding sequencein the wild-type AAV2 capsid we aimed to determine the contribution ofthese residues to the improved AAV-TT phenotype.

5×10¹⁰ vg of each mutant vector were injected in the lateral ventricleof P1 neonates. The animals were sacrificed 4 weeks post-injection andthe brains were dissected, sectioned and stained using an anti-GFPantibody. The results are shown in FIG. 33.

Interestingly, these data suggest that the AAV TT-S312N displaysenhanced transduction of mouse brain tissues as compared to the fullAAV2 TT vector. On the other hand, the amino acid changes S593A orD546E/G548D did not seem to affect the TT phenotype as similartransduction profiles could be observed throughout the brains. Whenobserving the stained sections at a higher magnification, thedifferences in transduction efficiency were further highlighted (FIG.34).

From the high magnification pictures, we could observe that the AAVTT-S312 seems to transduce neuronal tissues with higher efficiency thanthe original AAV-TT with 14 amino acid changes. In particular, we couldsee stronger transgene expression in the rostral side of the brain(cortex, striatum, hippocampus) after TT-S312N vector injection, both interms of level and of number of cells transduced. Despite the highvariability in injected neonatal brains due to the difficulty associatedwith targeting the injection site, this observation was confirmed in allthe animals analysed. On the other hand, the reversions S593A orD546E/G548D did not seem to have much impact on the TT vectortransduction phenotype.

Example 3 Targeted Amino Acid Mutations on the AAV2 True-Type Capsid,Selected from Results Obtained with the Mutant Combinations in Example 2

Based on the results from amino acid group mutations on the full AAV TTcapsid, we could determine that the mutation S312N seems to bebeneficial for the TT phenotype, further increasing its transductionefficiency in the brain. Furthermore, we observed that the reversionsS593A and D546G/G548E did not seem to affect the neuronal phenotype ofAAV-TT. We therefore hypothesised that the TT-specific residues S593,D546 and G548 could be excluded from the True-type capsid sequence,leaving instead the AAV2 WT residues at these positions to obtain afinal TT vector with only 10 amino acid changes.

In order to verify these hypotheses, we engineered theTT-S312N-D546G-G548D-S593A vector and tested its transduction efficiencyby neonatal mouse brain injections. Because the last neonateintracranial injections seemed to lead to a saturated signal in the GFPexpression detected, we decided to also inject the TT and the TT-S312Nvectors alongside this “pre-final” TT, using a 10 times lower dose thanused previously. By using this lower dose we aimed to avoid reachingsaturating levels of GFP staining in the transduced brains and avoiddifficulties in transduction efficiency comparison between differentmutants.

5×10⁰⁹ vg of each mutant vector were injected in the lateral ventricleof P1 neonates. The animals were sacrificed 4 weeks post-injection andthe brains were dissected, sectioned and stained using an anti-GFPantibody. The results are shown in FIG. 35.

As previously observed, these data suggest that the AAV TT-S312Ndisplays enhanced transduction of mouse brain tissues as compared to thefull AAV2 TT vector. On the other hand, the minimalTT-S312N-D546G/G548E-S593A vector did not seem to reach these higherlevels of transduction even though it also contained the internal S312Nmutation. This suggests that one or more of the amino acid changes playssome role in the TT phenotype. By reverting these residues back to AAV2WT equivalents, we lost some of the increased transduction ability. Whenobserving the stained sections at a higher magnification, theseobservations were confirmed (FIG. 36).

We decided to further investigate the transduction efficiency obtainedby each of these vectors by quantifying the total GFP expressionobtained in injected brains by enzyme-linked immunosorbent assay (ELISA)on full brain protein extracts. Briefly, 4 weeks after injection of5×10⁰⁹ vg of vectors, the animals were sacrificed, the whole brains wereharvest and lysed, and total brain proteins were extracted. Using a GFPprotein standard, we could then quantify the amounts of GFP proteinexpressed in each injected brain (FIG. 37).

We could confirm that the TT-S312N internal mutant transduces mousebrains with more efficiency than the full TT vector as it leads to moreGFP expression overall in all the injected brains. On the other hand theminimal TT vector, TT-S312N-D546G/G548E-S593A, seemed to lead to lowerlevels of transduction: although the average amount of GFP expressed perbrain seems higher on this graph, this was due to extreme GFP valuesmeasured in one of the brains as illustrated by the high error bar forthis condition. With this minimal TT vector, the variability betweenanimals was very high, with only one animal out of five performingbetter than the animals transduced with TT-S312N. This high variabilityled us to consider this provisional minimal TT vector with caution,especially since the immunohistochemistry analyses also showed that theTT-S312N variant performed better than the TT-S312N-DG-S593A.

We therefore selected the TT-S312N variant as our most preferred AAV TTvector, which is composed of the following 13 amino acid mutationscompared to the wild-type AAV2: V125I, V151A, A162S, T205S, Q457M,S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and A593S.

Although the above studies suggest the TT-S312N as the most preferredAAV-TT vector, these studies illustrate the individual function andcontribution of a number TT-specific residues. In particular, four aminoacids closely situated on the threefold spike of the capsid are likelyinvolved in receptor binding. In some embodiments, these residues arereverted in the AAV-TT back to the AAV2 WT corresponding residues. Forinstance, the mutations M457Q, A492S, D499E and Y533F may be engineeredon the AAV-TT capsid and this mutant vector analysed as previouslydescribed, in order to illustrate the role of these residues. In furtherembodiments, four amino acids situated in VP1/VP2 primary sequence,which are likely to be involved in trafficking of the virus, may bereverted in the AAV-TT back to AAV2 WT corresponding residues (I125V,A151V, S162A, S205T) and analysed similarly.

Example 4 Construction of Variant AAV Vectors in Other Serotypes

The function of the ttAAV2-specific amino acid changes in the context ofother, non-AAV2 serotypes can also be determined.

The capsid amino acid sequences of the main adeno-associated viruses(AAV), namely AAV1, 5, 6, 8, 9 and rh10, can be aligned with the onefrom ttAAV2, e.g. as shown in FIG. 9. This enables the identification ofwhich ttAAV2-specific amino acids are already present at the samepositions in other serotypes. The relevant residues in the variousserotypes are then mutated into the corresponding residues in wtAAV2.These changes demonstrate the importance of the ttAAV2-specific residuesfor the efficiency and biodistribution of each of the serotype.

As discussed above, the various mutant vectors, expressing GFP, are thensubmitted to a first screen by intracranial (IC) injection in CD1neonatal mice. The GFP signal obtained in the injected brains is thencompared to that obtained from the appropriate GFP-expressing serotypecontrols. The diminution or increase of GFP expression when mutating theidentified amino acids into their corresponding AAV2 residuesdemonstrates the importance of these particular residues at thesespecific positions. Where applicable, the injected brains are thenfurther sectioned and analyzed by immunohistochemistry. Additionally,chosen mutant serotypes are analyzed by intravenous (IV) injections intoneonatal mice in order to evaluate the biodistribution of the vectorsand compare it to the original, non-mutated counterparts.

In further embodiments, the relevant amino acids identified in thettAAV2 are inserted as key mutations into the other prominent serotypesat the relevant positions. The insertion of ttAAV2-specific residues inother AAV subtypes enables us to improve the transduction andbiodistribution profiles of each serotype.

Example 5 Modification of ttAAV2-Specific Residues Conserved in OtherAAV Serotypes Into the Corresponding AAV2 Residues

A comparative analysis of the capsid amino acid sequences of existingadeno-associated serotypes with the one from ttAAV2 first enabled us toidentify ttAAV2-specific residues that are conserved in other serotypes(see FIG. 9). These residues consist of S162, S205, S312, G548, S585 andT588. Each non-AAV2 serotype contains one or a combination of several ofthese residues at a corresponding amino acid position in its sequence.

In specific embodiments, each of these residues in the various serotypesare converted into the corresponding wild-type AAV2 amino-acid(s) andthe transduction efficiency of these new mutants is tested.

1) Modification of the AAV1 Serotype

AAV1 contains the residues S205, G549, S586 and T589 which correspond tothe following residues in ttAAV2: S205, G548, S585 and T588. When theVP1 monomer from AAV1 was aligned three-dimensionally with VP1 from AAV2we could verify that the corresponding residues in wtAAV2, namely T205,E548, R585 and R588, are at perfectly matching positions on the 3Dstructure (FIG. 21). We thus concluded that it would be significant toconvert each of the ttAAV2-specific residue(s) in AAV1 into thecorresponding wild-type AAV2 counterparts without affecting thethree-dimensional structure of the protein. In particular embodimentsthe following mutations are made in AAV1 capsid sequence: S205T, G549E,S586R, T589R.

2) Modification of the AAV5 Serotype

AAV5 contains the residues G537, S575 and T578 which correspond to thefollowing residues in ttAAV2: G548, S585 and T588. The R585 and R588residues in AAV2 are at matching positions with S575 and T578 in AAV5 onthe 3D structure. Although the residue E548 in AAV2 did not perfectlymatch the residue G537 in AAV5 according to the three-dimensionalstructure (FIG. 22), we still decided to include it in the study as bothresidues are relatively spatially close. Therefore in particularembodiments the following mutations are made in AAV5 capsid sequence:G537E, S575R, T578R.

3) Modification of the AAV6 Serotype

AAV6 contains the residues S205, G549, S586 and T589 which correspond tothe following residues in ttAAV2: S205, G548, S585 and T588. Thecorresponding residues in wtAAV2, namely T205, E548, R585 and R588 (FIG.23), are at perfectly matching positions on the VP1 3D structures.Therefore in particular embodiments the following mutations are made inAAV6 capsid sequence: S205T, G549E, S586R, T589R.

4) Modification of the AAV8 Serotype

AAV8 contains the residues S315 and T591 which correspond to thefollowing residues in ttAAV2: S312 and T588. The corresponding residuesin wtAAV2, namely N312 and R588, are at perfectly matching positions onthe VP1 3D structures (FIG. 24). Therefore in particular embodiments thefollowing mutations are made in AAV8 capsid sequence: S315N, T591R.

In one embodiment, the improved transduction efficiency imparted by theS312N mutation in TT AAV2 may be transferred to the AAV8 serotype byapplying the amino acid change S315N.

We mutated the AAV8 capsid sequence by site-directed mutagenesis andthereby created the AAV8-S315N vector plasmid. This plasmid was used toproduce recombinant AAV8-S315N vectors expressing an ITR-containingCMV-GFP transgene by double transfection of 293T cells. The vector wasthen purified from the cell lysate and from the harvested culturesupernatant by FPLC affinity chromatography, using an AVB sepharoseresin. The capsid titer and vector genome titer were assessed bySDS-PAGE and qPCR, respectively.

The mutant AAV8-S315N vector, expressing GFP, is screened by systemicinjections in CD1 neonatal mice. A titer-matched AAV8 vector is used asa control. GFP expression obtained in various organs after intra-jugularinjection of 2×10¹¹ vg is then analysed, primarily focusing on the liverwhere AAV8 has previously shown some strong transduction efficiency.

5) Modification of the AAV9 Serotype

AAV9 contains the residues S162, S205, G549 and S586 which correspond tothe following residues in ttAAV2: S162, S205, G548 and S585. Thecorresponding residues in AAV2, namely A162, T205, E548 and R585, are atperfectly matching positions on the VP1 3D structures (FIG. 25).Therefore in particular embodiments the following mutations are made inAAV8 capsid sequence: S162A, S205T, G549E, and S586R.

6) Modification of the AAVrh10 Serotype

AAVrh10 contains the residue G551 which corresponds with G548 withttAAV2. Considering how conserved this residue and position appearsamong the various serotypes, we assume that G551 in AAVrh10 will alignwith E548 in wtAAV2 three-dimensionally. Therefore in one embodiment thefollowing mutation is made in AAVrh10 capsid sequence: G551E.

7) Modification of the AAV3B and AAV-LK03 Serotypes

Similarly to the AAV8 serotype, we observed that the AAV3B serotype alsocontains an internal serine at position 312 after aligning the capsidprotein VP1 sequence with the one from AAV-TT (see AAV3B capsid sequencein FIG. 38).

The newly described LK03 AAV vector, a chimeric capsid composed of fivedifferent parental AAV capsids engineered by M. A. Kay by DNA-shuffling,also contains the residue S312 in the internal side of the capsid (seeLisowski et al., Selection and evaluation of clinically relevant AAVvariants in a xenograft liver model, Nature 506, 382-386 (2014)). Thecapsid sequence of AAV-LK03 is disclosed in WO 2013/029030 and shown inFIG. 39.

Therefore in further embodiments, the AAV3B and the AAV-LK03 vectors aremutated by applying the amino-acid change S312N in both vectors. Thesenew mutants, and their corresponding AAV control serotypes, are alsotested by intra-jugular injections in neonatal mice. The GFP expressionobtained in various harvested organs is then analysed.

Example 6 Identification of ttAAV2-Specific Amino Acids That areTransferable Between AAV Serotypes

In further embodiments, the key amino acids identified during the ttAAV2characterization are inserted into the other prominent serotypes at therelevant positions. The newly engineered vectors are then tested usingthe appropriate non-mutated serotypes as controls. This validates theimportance of individual amino acids at specific positions on AAVcapsids, independently of the serotype.

1) Residues S585, T588, S312, D546, G548 and S593

AAV1, AAV5 and AAV6 naturally contain the same amino acid residue atpositions in their capsid protein sequences corresponding to G548, S585and T588 in ttAAV2. Therefore in further embodiments, the capsidproteins in these serotypes are mutated at matching positions to includethe other residues S312, D546, and S593 present in ttAAV2. SimilarlyAAV8, which already contains the same amino acid residue as in ttAAV2 atpositions corresponding to S312 and T588 in ttAAV2, is further mutatedto contain residues corresponding to S585, D546, G548 and S593 inttAAV2. AAV9, that already contains corresponding residues to G548 andS585 in ttAAV2, is mutated to include residues corresponding to T588,S312, D546 and S593 in ttAAV2. Finally AAV10, which already contains aresidue corresponding to G548, is further modified to also containresidues corresponding to S585, T588, S312, G548 and S593.

2) Residues I125, A151, S162, S205, M457, A492, D499 and Y533

AAV1, and AAV6 already naturally contain the residue S205. Therefore infurther embodiments these serotypes are mutated at positionscorresponding to the residues I125, A151, 5162, M457, A492, D499 andY533 in ttAAV2. Similarly AAV9, that already contains the residues S162and S205, is further mutated to contain residues corresponding to I125,A151, M457, A492, D499 and Y533 in ttAAV2. Finally, AAV5, 8 and 10 aremodified to display residues corresponding to I125, A151, S162, S205,M457, A492, D499 and Y533 in ttAAV2.

The positions the mutations present in ttAAV2 and the correspondingresidues present in the wild type capsid protein VP1 sequences of otherAAV serotypes are shown in Table 1 below. In general, variant non-AAV2vectors can be constructed by mutating any of the residues shown inTable 1 for these serotypes. The residues shown in italics are residueswhich are already present in ttAAV2 at a corresponding position. Inpreferred embodiment, the non-AAV2 serotypes are mutated at one or morethe residues shown in non-italic script. In this ways, the advantageousproperties shown by ttAAV2 can be transferred into alternative AAVserotypes.

TABLE 1 ttAAV2 AAV1 AAV5 AAV6 AAV8 AAV9 AAV10 I125 V125 V124 V125 V125L125 V125 A151 Q151 K150 Q151 Q151 Q151 Q151 S162 T162 K153 T162 K163S162 K163 S205 S205 A195 S205 A206 S205 A206 S312 N313 R303 N313 S315N314 N315 M457 N458 T444 N458 T460 Q458 T460 A492 K493 S479 K493 T495V493 L495 D499 N500 V486 N500 N502 E500 N502 Y533 F534 T520 F534 F536F534 F536 D546 S547 P533 S547 N549 G547 G549 G548 G549 G537 G549 A551G549 G551 S585 S586 S575 S586 Q588 S586 Q588 T588 T589 T578 T589 T591A589 A591 S593 G594 G583 G594 G596 G594 G596

Further Embodiments of the Invention

The invention also relates additional aspects, as defined in thefollowing summary paragraphs:

1. A recombinant adeno-associated virus (AAV) vector comprising;

(a) a variant AAV capsid protein, wherein the variant AAV capsid proteincomprises at least one amino acid substitution with respect to a wildtype AAV capsid protein; wherein the at least one amino acidsubstitution is present at a position corresponding to one or more ofthe following positions in an AAV2 capsid protein sequence: 125, 151,162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; and

(b) a heterologous nucleic acid comprising a nucleotide sequenceencoding a gene product.

2. A recombinant AAV vector according to paragraph 1, wherein (i) thevector comprises a variant AAV2 capsid protein; (ii) the variant AAVcapsid protein comprises a sequence of SEQ ID NO:2, or a sequence havingat least 95% sequence identity thereto; (iii) the wild type AAV capsidprotein is from AAV2; and/or (iv) the wild type AAV capsid proteincomprises a sequence of SEQ ID NO:1.

3. A recombinant AAV vector according to paragraph 2, wherein thevariant AAV2 capsid protein comprises one or more of the followingresidues: I125, A151, S162, S205, S312, M457, A492, D499, Y533, D546,G548, S585, T588 and/or S593.

4. A recombinant AAV vector according to paragraph 2 or paragraph 3,wherein the variant AAV2 capsid protein comprises one or more of thefollowing amino acid substitutions with respect to a wild type AAV2capsid protein: V125I, V151A, A162S, T205S, N312S, Q457M, S492A, E499D,F533Y, G546D, E548G, R5855, R588T and/or A593S.

5. A recombinant AAV vector according to any of paragraphs 1 to 3,wherein the variant AAV capsid protein is from AAV1, AAV5, AAV6, AAV8,AAV9 or AAV10.

6. A recombinant AAV vector according to paragraph 5, wherein (i) thevector comprises a variant AAV1 capsid protein, (ii) the variant AAVcapsid protein comprises a sequence having at least 95% sequenceidentity to SEQ ID NO:3; (iii) the wild type AAV capsid protein is fromAAV1; and/or (iv) the wild type AAV capsid protein comprises a sequenceof SEQ ID NO:3;

and wherein at least one amino acid substitution is present at one ormore of the following positions in the AAV1 capsid protein sequence:125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or594.

7. A recombinant AAV vector according to paragraph 6, wherein thevariant AAV1 capsid protein comprises one or more of the following aminoacid substitutions with respect to a wild type AAV1 capsid protein:

(a) V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D,and/or G594S; and/or

(b) S205T, G549E, S586R and/or T589R.

8. A recombinant AAV vector according to paragraph 5, wherein (i) thevector comprises a variant AAV5 capsid protein, (ii) the variant AAVcapsid protein comprises a sequence having at least 95% sequenceidentity to SEQ ID NO:4; (iii) the wild type AAV capsid protein is fromAAV5; and/or (iv) the wild type AAV capsid protein comprises a sequenceof SEQ ID NO:4;

and wherein at least one amino acid substitution is present at one ormore of the following positions in the AAV5 capsid protein sequence:124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578 and/or583.

9. A recombinant AAV vector according to paragraph 8, wherein thevariant AAV5 capsid protein comprises one or more of the following aminoacid substitutions with respect to a wild type AAV5 capsid protein:

(a) V124I, K150A, K153S, A195S, R303S, T444M, S479A, V486D, T520Y,P533D, and/or G583S; and/or

(b) G537E, S575R and/or T578R.

10. A recombinant AAV vector according to paragraph 5, wherein (i) thevector comprises a variant AAV6 capsid protein, (ii) the variant AAVcapsid protein comprises a sequence having at least 95% sequenceidentity to SEQ ID NO:5; (iii) the wild type AAV capsid protein is fromAAV6; and/or (iv) the wild type AAV capsid protein comprises a sequenceof SEQ ID NO:5;

and wherein at least one amino acid substitution is present at one ormore of the following positions in the AAV6 capsid protein sequence:125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or594.

11. A recombinant AAV vector according to paragraph 10, wherein thevariant AAV6 capsid protein comprises one or more of the following aminoacid substitutions with respect to a wild type AAV6 capsid protein:

(a) V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D,and/or G594S; and/or

(b) S205T, G549E, S586R and/or T589R.

12. A recombinant AAV vector according to paragraph 5, wherein (i) thevector comprises a variant AAV8 capsid protein, (ii) the variant AAVcapsid protein comprises a sequence having at least 95% sequenceidentity to SEQ ID NO:6; (iii) the wild type AAV capsid protein is fromAAV8; and/or (iv) the wild type AAV capsid protein comprises a sequenceof SEQ ID NO:6;

and wherein at least one amino acid substitution is present at one ormore of the following positions in the AAV8 capsid protein sequence:125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and/or596.

13. A recombinant AAV vector according to paragraph 12, wherein thevariant AAV8 capsid protein comprises one or more of the following aminoacid substitutions with respect to a wild type AAV8 capsid protein:

(a) V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D,A551G, Q588S and/or G596S; and/or

(b) S315N and/or T591R.

14. A recombinant AAV vector according to paragraph 5, wherein (i) thevector comprises a variant AAV9 capsid protein, (ii) the variant AAVcapsid protein comprises a sequence having at least 95% sequenceidentity to SEQ ID NO:7; (iii) the wild type AAV capsid protein is fromAAV9; and/or (iv) the wild type AAV capsid protein comprises a sequenceof SEQ ID NO:7;

and wherein at least one amino acid substitution is present at one ormore of the following positions in the AAV9 capsid protein sequence:125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589 and/or594.

15. A recombinant AAV vector according to paragraph 14, wherein thevariant AAV9 capsid protein comprises one or more of the following aminoacid substitutions with respect to a wild type AAV9 capsid protein:

(a) L125I, Q151A, N314S, Q458M, V493A, E500D, F534Y, G547D, A589T and/orG594S; and/or

(b) S162A, S205T, G549E and/or S586R.

16. A recombinant AAV vector according to paragraph 5, wherein (i) thevector comprises a variant AAVrh10 capsid protein, (ii) the variant AAVcapsid protein comprises a sequence having at least 95% sequenceidentity to SEQ ID NO:8; (iii) the wild type AAV capsid protein is fromAAVrh10; and/or (iv) the wild type AAV capsid protein comprises asequence of SEQ ID NO:8;

and wherein at least one amino acid substitution is present at one ormore of the following positions in the AAV10 capsid protein sequence:125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and/or596.

17. A recombinant AAV vector according to paragraph 16, wherein thevariant AAVrh10 capsid protein comprises one or more of the followingamino acid substitutions with respect to a wild type AAVrh10 capsidprotein:

(a) V125I, Q151A, K163S, A206S, N315S, T460M, L495A, N502D, F536Y,G549D, Q588S, A591T and/or G596S; and/or

(b) G551E.

18. A recombinant AAV vector according to any preceding paragraph,wherein the recombinant AAV vector exhibits increased transduction of aneuronal or retinal tissue compared to an AAV vector comprising acorresponding wild type AAV capsid protein.

19. A recombinant AAV vector according to any preceding paragraph,wherein the gene product comprises an interfering RNA or an aptamer.

20. A recombinant AAV vector according to any of paragraphs 1 to 18,wherein the gene product comprises a polypeptide.

21. A recombinant AAV vector according to paragraph 20, wherein the geneproduct comprises a neuroprotective polypeptide, an anti-angiogenicpolypeptide, or a polypeptide that enhances function of a neuronal orretinal cell.

22. A recombinant AAV vector according to paragraph 21, wherein the geneproduct comprises glial derived neurotrophic factor, fibroblast growthfactor, nerve growth factor, brain derived neurotrophic factor,rhodopsin, retinoschisin, RPE65 or peripherin.

23. A pharmaceutical composition comprising:

(a) a recombinant AAV vector according to any preceding paragraph; and

(b) a pharmaceutically acceptable excipient.

24. A method for delivering a gene product to a neuronal or retinaltissue in a subject, the method comprising administering to the subjecta recombinant AAV vector or pharmaceutical composition according to anypreceding paragraph.

25. A method for treating a neurological or ocular disorder, the methodcomprising administering to the subject a recombinant AAV vector orpharmaceutical composition according to any preceding paragraph.

26. A recombinant AAV vector or pharmaceutical composition according toany of paragraphs 1 to 23, for use in treating a neurological or oculardisorder.

27. A method, recombinant AAV vector or pharmaceutical composition foruse according to any of paragraphs 24 to 26, wherein the neurologicaldisorder is a neurodegenerative disease.

28. A method, recombinant AAV vector or pharmaceutical composition foruse according to any of paragraphs 24 to 26, wherein the ocular disorderis glaucoma, retinitis pigmentosa, macular degeneration, retinoschisisor diabetic retinopathy.

29. An isolated variant AAV capsid protein, wherein the variant AAVcapsid protein comprises at least one amino acid substitution withrespect to a wild type AAV capsid protein; wherein the at least oneamino acid substitution is present at one or more of the followingpositions in an AAV2 capsid protein sequence: 125, 151, 162, 205, 312,457, 492, 499, 533, 546, 548, 585, 588 and/or 593; or at one or morecorresponding positions in an alternative AAV capsid protein sequence.

30. An isolated nucleic acid comprising a nucleotide sequence thatencodes a variant AAV capsid protein as defined in paragraph 29.

31. An isolated host cell comprising a nucleic acid as defined inparagraph 30.

1. A recombinant adeno-associated virus (AAV) vector comprising: (a) avariant AAV2 capsid protein, wherein the variant AAV2 capsid proteincomprises at least four amino acid substitutions with respect to a wildtype AAV2 capsid protein; wherein the at least four amino acidsubstitutions are present at the following positions in an AAV2 capsidprotein sequence: 457, 492, 499 and 533; and (b) a heterologous nucleicacid comprising a nucleotide sequence encoding a gene product.
 2. Arecombinant AAV vector according to claim 1, wherein (i) the variant AAVcapsid protein comprises a sequence of SEQ ID NO:2, or a sequence havingat least 95% sequence identity thereto; and/or (ii) the wild type AAVcapsid protein comprises a sequence of SEQ ID NO:1.
 3. A recombinant AAVvector according to claim 1 or claim 2, wherein the variant AAV2 capsidprotein comprises one or more of the following residues: M457, A492,D499 and Y533.
 4. A recombinant AAV vector according to claim 2 or claim3, wherein the variant AAV2 capsid protein comprises one or more of thefollowing amino acid substitutions with respect to a wild type AAV2capsid protein: Q457M, S492A, E499D and F533Y.
 5. A recombinant AAVvector according to any of claims 1 to 4, wherein the variant AAV2capsid protein further comprises one or more amino acid substitutionswith respect to the wild type AAV capsid protein at the followingpositions in the AAV2 capsid protein sequence: 125, 151, 162 and
 205. 6.A recombinant AAV vector according to claim 5, wherein the variant AAV2capsid protein comprises one or more of one or more of the followingresidues: I125, A151, S162 and S205.
 7. A recombinant AAV vectoraccording to claim 5 or claim 6, wherein the variant AAV2 capsid proteincomprises one or more of the following amino acid substitutions withrespect to a wild type AAV2 capsid protein: V125I, V151A, A162S andT205S.
 8. A recombinant AAV vector according to any of claims 1 to 7,wherein the variant AAV2 capsid protein further comprises one or moreamino acid substitutions with respect to the wild type AAV capsidprotein at the following positions in the AAV2 capsid protein sequence:585 and
 588. 9. A recombinant AAV vector according to claim 8, whereinthe variant AAV2 capsid protein comprises one or more of one or more ofthe following residues: S585 and T588.
 10. A recombinant AAV vectoraccording to claim 8 or claim 9, wherein the variant AAV2 capsid proteincomprises one or more of the following amino acid substitutions withrespect to a wild type AAV2 capsid protein: R585S and R588T.
 11. Arecombinant AAV vector according to any of claims 1 to 10, wherein thevariant AAV2 capsid protein further comprises one or more amino acidsubstitutions with respect to the wild type AAV capsid protein at thefollowing positions in the AAV2 capsid protein sequence: 546, 548 and593.
 12. A recombinant AAV vector according to claim 11, wherein thevariant AAV2 capsid protein comprises one or more of one or more of thefollowing residues: D546, G548, and S593.
 13. A recombinant AAV vectoraccording to claim 11 or claim 12, wherein the variant AAV2 capsidprotein comprises one or more of the following amino acid substitutionswith respect to a wild type AAV2 capsid protein: G546D, E548G and A593S.14. A recombinant AAV vector according to any of claims 1 to 13, whereinthe variant AAV2 capsid protein comprises the residue N312.
 15. Arecombinant adeno-associated virus (AAV) vector comprising: (a) avariant AAV8 capsid protein, wherein the variant AAV8 capsid proteincomprises an amino acid substitution with respect to a wild type AAV8capsid protein at position 315 in an AAV8 capsid protein sequence; and(b) a heterologous nucleic acid comprising a nucleotide sequenceencoding a gene product.
 16. A recombinant AAV vector according to claim15, wherein (i) the variant AAV capsid protein comprises a sequencehaving at least 95% sequence identity to SEQ ID NO:6; and/or (ii) thewild type AAV capsid protein comprises a sequence of SEQ ID NO:6.
 17. Arecombinant AAV vector according to claim 15 or claim 16, wherein thevariant AAV8 capsid protein comprises the amino acid substitution S315Nwith respect to a wild type AAV8 capsid protein.
 18. A recombinant AAVvector according to any of claims 15 to 17, further comprising one ormore amino acid substitution present at one or more of the followingpositions in the AAV8 capsid protein sequence: 125, 151, 163, 206, 460,495, 502, 536, 549, 551, 588, 591 and/or
 596. 19. A recombinant AAVvector according to claim 18, wherein the variant AAV8 capsid proteincomprises one or more of the following amino acid substitutions withrespect to a wild type AAV8 capsid protein: (a) V125I, Q151A, K163S,A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and/or G596S;and/or (b) T591R.
 20. A recombinant adeno-associated virus (AAV) vectorcomprising: (a) a variant AAV3B capsid protein, wherein the variantAAV3B capsid protein comprises an amino acid substitution with respectto a wild type AAV3B capsid protein at position 312 in an AAV3B capsidprotein sequence; and (b) a heterologous nucleic acid comprising anucleotide sequence encoding a gene product.
 21. A recombinant AAVvector according to claim 20, wherein (i) the variant AAV3B capsidprotein comprises a sequence having at least 95% sequence identity toSEQ ID NO:11; and/or (ii) the wild type AAV capsid protein comprises asequence of SEQ ID NO:11.
 22. A recombinant AAV vector according toclaim 20 or claim 21, wherein the variant AAV3B capsid protein comprisesthe amino acid substitution S312N with respect to a wild type AAV3Bcapsid protein.
 23. A recombinant adeno-associated virus (AAV) vectorcomprising: (a) a variant AAV-LK03 capsid protein, wherein the variantAAV-LK03 capsid protein comprises an amino acid substitution at position312 with respect to a AAV-LK03 capsid protein sequence as defined in SEQID NO:12; and (b) a heterologous nucleic acid comprising a nucleotidesequence encoding a gene product.
 24. A recombinant AAV vector accordingto claim 23, wherein the variant AAV-LK03 capsid protein comprises asequence having at least 95% sequence identity to SEQ ID NO:12.
 25. Arecombinant AAV vector according to any of claims 1 to 14, wherein therecombinant AAV vector exhibits increased transduction of a neuronal orretinal tissue compared to an AAV vector comprising a corresponding wildtype AAV capsid protein.
 26. A recombinant AAV vector according to anyof claims 15 to 24, wherein the recombinant AAV vector exhibitsincreased transduction of liver tissue compared to a corresponding wildtype AAV capsid protein.
 27. A recombinant AAV vector according to anypreceding claim, wherein the gene product comprises an interfering RNAor an aptamer.
 28. A recombinant AAV vector according to any of claims 1to 27, wherein the gene product comprises a polypeptide.
 29. Arecombinant AAV vector according to claim 28, wherein the gene productcomprises a neuroprotective polypeptide, an anti-angiogenic polypeptide,or a polypeptide that enhances function of a neuronal or retinal cell.30. A recombinant AAV vector according to claim 29, wherein the geneproduct comprises glial derived neurotrophic factor, fibroblast growthfactor, nerve growth factor, brain derived neurotrophic factor,rhodopsin, retinoschisin, RPE65 or peripherin.
 31. A pharmaceuticalcomposition comprising: (a) a recombinant AAV vector according to anypreceding claim; and (b) a pharmaceutically acceptable excipient.
 32. Amethod for delivering a gene product to a tissue in a subject, themethod comprising administering to the subject a recombinant AAV vectoror pharmaceutical composition according to any preceding claim.
 33. Amethod according to claim 32, wherein the tissue is selected from blood,bone marrow, muscle tissue, neuronal tissue, retinal tissue, pancreatictissue, liver tissue, kidney tissue, lung tissue, intestinal tissue orheart tissue.
 34. A method according to claim 33, wherein the tissue isneuronal, retinal or liver tissue.
 35. A method for treating a disorderin a subject, the method comprising administering to the subject arecombinant AAV vector or pharmaceutical composition according to anypreceding claim.
 36. A recombinant AAV vector or pharmaceuticalcomposition according to any of claims 1 to 31, for use in treating adisorder in a subject.
 37. A method, recombinant AAV vector orpharmaceutical composition for use according to claim 35 or claim 36,wherein the disorder is a neurological, ocular or hepatic disorder. 38.A method, recombinant AAV vector or pharmaceutical composition for useaccording to claim 37, wherein the neurological disorder is aneurodegenerative disease.
 39. A method, recombinant AAV vector orpharmaceutical composition for use according to claim 37, wherein theocular disorder is glaucoma, retinitis pigmentosa, macular degeneration,retinoschisis or diabetic retinopathy.